Daphnia magna and Ceriodaphnia dubia have similar sensitivity in standard acute and chronic toxicity tests

Abstract

The cladocerans Daphnia magna and Ceriodaphnia dubia have been used for decades to assess the hazards of chemicals and effluents but toxicity data for these species have traditionally been treated separately. Numerous standard acute and chronic test guidelines have been developed for both species. In this study, data were compiled and curated for acute survival (48h) and growth and reproduction tests with D. magna (21d chronic) and C. dubia (7d chronic) toxicity assays. Orthogonal regressions were developed to statistically compare the acute and chronic sensitivity of D. magna and C. dubia across a diversity of chemicals and modes of action. Acute orthogonal regressions between D. magna and D. pulex, a widely accepted surrogate species, were used to set a data-driven benchmark for what would constitute a suitable D. magna surrogate. The results indicate there is insufficient evidence to suggest a difference in acute or chronic sensitivity of D. magna and C. dubia in standard toxicity tests. Further, the variability in the acute D. magna and C. dubia regressions were of the same magnitude as D. magna and D. pulex regressions. Slope and y-intercept values were also comparable. The absence of significant differences in toxicity values suggest similar species sensitivity in standard tests across a range chemical classes and MOA.

INTRODUCTION

Environmental risk assessments (ERA) are used to describe the likelihood a chemical will be present in the environment at a concentration capable of eliciting an adverse impact on ecological structure or function. Biological effects can be measured using a variety of techniques varying in technical complexity, cost, and biological realism. At the lowest tiers, quantitative structure activity relationships (QSAR) modelling approaches (e.g., ECOSAR) can be used to estimate aquatic toxicity based on physical/chemical properties. At the highest tiers, complex approaches such as mesocosms and species sensitivity distributions (SSDs) can be used to characterize environmental hazard with greater certainty (Shaw and Kennedy, 1996; Versteeg et al., 1999). Assessment factors are then applied to the most sensitive measured endpoint to derive a Predicted No Effect Concentration (PNEC); with small assessment factors used for high-tiered data and large assessment factors when data is limited. Assessment factors vary based on regulatory jurisdictions and can influence the overall interpretation of a chemical’s safety profile (Belanger et al., 2021).

In practice, ERAs for industrial compounds rely heavily on single species toxicity tests using standard freshwater test organisms and standardized experimental test guidelines. Three trophic levels of aquatic organisms are typically examined: algae, invertebrates, and fish. Philosophically these represent important links within a simplified food chain (primary producer, primary consumer, and secondary consumer, respectively). Standard model organisms in use today were established through a combination of practical considerations and serendipity. Good model organisms must be easy to culture, have short life cycles, a well understood life history, require small testing volumes, and be sensitive to a broad range of aquatic contaminants. Ideally, these species would also fulfill an important ecological niche and have broad geographical distribution.

Aquatic invertebrates play important and varied roles within an ecological community. This trophic level encompasses a wide diversity of organisms with unique morphology, feeding strategies (e.g., shredders, grazers, filter-feeders), and ecological niches. Aquatic invertebrates used in toxicity tests encompass a diversity of taxa including daphnids, amphipods, crayfish, stoneflies, mayflies, midges, snails, and rotifers. For standard regulatory applications, Daphnia magna (and to a lesser extent, Daphnia pulex) are overwhelming the most tested species (Fairbrother et al., 2014). This is largely due to ease of culture, availability of standardized methods, and explicit inclusion in regulatory frameworks (e.g., REACH).

D. magna, D. pulex and C. dubia belong to the phylogenetic family Daphniidae. All three species are filter-feeding, pelagic zooplankton that play common roles in freshwater planktonic communities and reside in similar geographical locations (Versteeg et al., 1997). Reproductive strategies are also identical: under low-stress conditions typical of standard laboratory cultures, neonates are created through asexual parthenogenesis where high-stress conditions can result in the production of ephippia and/or male neonates. D. magna are the largest of the three organisms. This larger size corresponds with a longer time to reach reproductive maturity and longer generation times (Table 1). Adult C. dubia are roughly one sixth the size of D. magna. C. dubia reach reproductive maturity in ∼3 days. With their shorter generation time, a three-brood chronic toxicity assay can be completed with C. dubia in approximately 5 to 8 days.

Table 1.

Comparison of life history and ecological traits of Daphnia magna, Daphnia pulex, and Ceriodaphnia dubia species

	Daphnia magna	Daphnia pulex	Ceriodaphnia dubia
Habitat Preference	Lakes, ponds, streams	Lakes, ponds, streams	Lakes, ponds, marshes, slow water, littoral zones
Geographic Distribution	North America	North America, Europe	Europe, Asia, North America
Adult size	5–6 mm	3 mm	∼0.9 mm
Time to maturity	6–10 d	∼6–8 d	∼3 d
Generation time	∼4–6 d	∼2–4 d	2 d
Clutch size	4–25 neonates	2–15 neonates	5–15 neonates
Preferred pH	6.5–8.5	6.5–8.5	6.5–8.5
Optimal Temperature	20 ± 2 C	20 ± 2 C	25 ± 1 C
Food	Algae	Algae	Algae, YCT

Daphnia species, particularly D. pulex and D. magna, have been the most common model freshwater invertebrate species since the 1960s, and have been recognized as a useful test organism as far back as 1945 (Anderson, 1944). Daphnia magna chronic toxicity testing protocols, dating back to at least 1971, were developed at industry, government and contract laboratories (P&G archival information for protocols at EG&G Bionomics, 1971; (Biesinger and Christensen, 1972); see also (Maki, 1979)). By the 1970’s, test standardization organizations (ASTM, US EPA, ISO, OECD) began to codify the use of daphnids in guideline forms based almost exclusively on D. magna (Persoone et al., 2009). Early acute test guidelines at the international level commonly employed 24 h exposure periods with an option for extension to 48 h (OECD, 1984); however, current guidelines rely exclusively on the 48 h duration for acute toxicity (OECD, 2004). D. pulex is used within the US, but primarily for effluent toxicity testing and not for chemical hazard assessments.

The use of D. magna in chronic toxicity tests began to emerge in the 1970’s and international guidelines for daphnid reproduction and survival tests ranged from 14 to 21 d in duration (OECD, 1984, 2012), however test methods eventually converged on a 21d duration. When the US whole effluent toxicity testing program was developed, it was rare for Daphnia chronic tests to be conducted on effluents due to the ephemeral nature of discharges and need for long exposures. The alternative daphnid chronic toxicity test, using Ceriodaphnia dubia, which provides 3 broods in 7 days and of sufficient number to be statistically sensitive was developed to fulfill the need for effluent testing and at a lower cost (due to testing frequency) (Mount and Norberg, 1984). ISO adopted a test guideline (ISO, 2017) closely following that of US EPA (2002a). By 2012, OECD had also officially included C. dubia as an optional cladoceran test species although in practice the adoption has been greatest in US, Canada and Japan and substantially less so in Europe (Versteeg et al., 1997).

The reluctance to adopt C. dubia versus D. magna may be tied to a number of issues: (1) Early on (1983–1990; (Berner, 1987)), Ceriodaphnia taxonomy was not as clear to non-taxonomy experts and a range of species other than C. dubia including the morphologically similar C. reticulata and C. affinis were identified, leading to confusion as to what was tested at the time and a lower confidence that the species named was indeed correct; (2) Ecotoxicologists questioned the applicability of a shorter exposure duration for Ceriodaphnia versus Daphnia especially for hydrophobic substances; (3) Questions regarding test repeatability in the context of both effluents and chemicals; (4) Overall regulatory comfort for a less well studied species (in the beginning) to act as an equivalent surrogate for invertebrates was also present. Each of these issues have been mitigated over time as information has accumulated (Versteeg et al., 1997). The most substantive issues are those of repeatability and equivalency in sensitivity. Regarding repeatability, numerous studies have documented that the Ceriodaphnia assay repeatability is consistently in the range of most ecotoxicity assays (20–40%) depending on the statistical endpoint and study being considered (USEPA 2002c; Moore et al., 2000). The main remaining issue is that of equivalency in sensitivity which is further addressed in the present research.

In 1997, Versteeg et al compared the sensitivity of C. dubia and D. magna. Acute toxicity data from 42 different compounds (30 organics, 12 inorganics) and 6 different effluents were compared (Versteeg et al., 1997). C. dubia were more sensitive than D. magna 73% of the time by an average factor of 2.4. The toxicity to both species was highly correlated (r²=0.96) with a linear regression slope of approximately 1. A comparison of chronic toxicity was also conducted, based on 16 compounds (12 organic, 4 inorganic) and 4 effluents. A significant correlation in chronic toxicity values was observed (r²=0.62) and no statistical difference was found between species. These observations suggest that C. dubia could be used to predict the acute and chronic toxicity of D. magna. However, these comparisons were derived from a relatively small number of chemicals with limited experimental replication. Large databases, such as the EnviroTox database (Connors et al., 2019), can be used to further explore the inherent sensitivity differences between D. magna and C. dubia for more substances with a wider diversity of chemicals.

Data requirements, including the required test methodology, can vary by regulatory jurisdiction. The use of a species not explicitly outlined in the test methodology can result in study rejection. In regulatory applications, D. pulex is widely accepted as a substitute for D. magna without restrictions. While not explicitly stated, this implies these species are equi-sensitive and any variation in species sensitivity between D. magna and D. pulex, be it experimental and/or biological variation, is not of concern for industrial chemical assessments. Here, we critically evaluate standard acute and chronic testing methodologies for D. magna, D. pulex, and C. dubia to identify key differences that may influence test interpretation. In order to determine the comparative sensitivity, we first examine the relative acute sensitivity of D. magna and D. pulex. The results from these orthogonal regressions will be used to set a data-driven benchmark for what would constitute a suitable D. magna surrogate. The slope and y-intercept of the relationship is then contrasted with the D. magna and C. dubia sensitivity relationship. Chronic toxicity comparisons were also completed. Collectively, this information was used to statistically evaluate the relative sensitivity of these aquatic invertebrates and determine whether C. dubia and D. magna toxicity results could be considered interchangeable across a range of chemical classes and MOA.

METHODS

Comparison of standard test methods

A selection of standard acute and chronic invertebrate test guidelines were identified for critical review. Key information related to study design, water quality requirements, experimental and statistical endpoints, and validity criteria were summarized. Species-specific requirements were identified and summarized.

Data compilation

Daphnia magna, Daphnia pulex, and Ceriodaphnia dubia acute and chronic toxicity data were compiled from the EnviroTox database v1.3 ((HESI), 2019) and supplemented with 30 internal studies conducted at Procter & Gamble. The EnviroTox database is a publicly available repository of environmental effects data. This database is an amalgamation of numerous data sources (e.g., US EPA ECOTOX, USEPA Pesticide data, METI, USGS Columbia, peer-reviewed literature, etc). Individual records contained within the EnviroTox database have not necessarily been quality assured. This may result in the inclusion of some poorer-quality data (for more information, see (Connors et al., 2019)). Both nominal and analytically measured exposure concentrations are contained within the database. Due to limitations in data reporting, it is not possible to separate the data based on this attribute. All studies within the database have been systematically classified as acute or chronic based on study duration, experimental endpoint, and statistical endpoint (Connors et al., 2019). These classifications were utilized to extract relevant studies. Metals and ammonia were excluded from further analysis due to inconsistent toxicity normalization within the EnviroTox database (e.g., tests conducted with varying dilution water parameters that may influence toxicity interpretation). Additional manual curation was completed to remove suspected duplicate entries, as identified by matching CAS, species, test statistic and effect values. Consensus MOA assignments were also extracted for further analysis (Kienzler et al., 2019). All studies were presumed to be conducted under standard test guideline conditions (e.g., pH, temperature, water hardness) relevant for each species.

Separately, the highly curated acute toxicity values used to develop the US EPA web-based Interspecies Correlation Estimation (WebICE) models were accessed (Raimondo et al., 2015). These data have undergone standardization and manual quality assurance/quality control measures by the US EPA Office of Research and Development to assure studies meet regulatory guidelines for organism age, study duration, test design, and water quality characteristics. Because toxicity normalization procedures were evenly applied, metals and ammonia studies from WebICE were included in the data compilation. This database includes high quality studies conducted nominally or with analytically verified concentrations.

In both data sources, acute toxicity values were obtained from a 48h mortality assays with effect concentrations reported as LC/EC50 values. Data sourced from WebICE represented a relatively small number of chemicals (20–30 chemicals; Table 4) and represent a fairly narrow range of chemical diversity and physical/chemical properties. EnviroTox contains considerably more paired acute toxicity data, however, this data has not undergone the same level of manual curation as WebICE. A total of 125 chemicals (1197 studies) were identified with acute toxicity data for both D. magna and D. pulex and 145 chemicals (1375 studies) had data for both D. magna and C. dubia. Many chemicals had multiple reported EC50 values for a given chemical and species combination. Relatively few chemicals were contained in both the EnviroTox and WebICE databases: 9 chemicals with D. magna and D. pulex data, and 13 chemicals with D. magna and C. dubia data.

Table 4.

Daphnia magna, Daphnia pulex, Ceriodaphnia dubia orthogonal regression summary. All hazard units are denoted in µg/L units.

Comparison	Test Type	Data	# Entries	# Chems	Equation	Slope 95% CI	Intercept 95% CI	r	MSE
D. magna, D. pulex	Acute	WebICE	78	21	LC50DM = 0.967 × LC50DP + 0.165	0.89, 1.05	−0.04, 0.37	0.99	0.11
		EnviroTox	1,197	125	LC50DM = 0.928 × LC50DP + 0.470	0.84, 1.02	0.15, 0.79	0.87	0.72
		EnviroTox w. reps	827	50	LC50DM = 0.918 × LC50DP + 0.446	0.83, 1.01	0.15, 0.74	0.95	0.30
D. magna, C. dubia	Acute	WebICE	258	32	LC50DM = 0.971 × LC50CD + 0.250	0.90, 1.05	−0.03, 0.54	0.98	0.24
		EnviroTox	1,375	145	LC50DM = 0.908 × LC50CD + 0.611	0.86, 0.96	0.43, 0.80	0.95	0.32
		EnviroTox w. reps	872	50	LC50DM = 0.891 × LC50CD + 0.688	0.82, 0.96	0.43, 0.94	0.97	0.27
D. magna, C. dubia	Chronic	EnviroTox	229	51	NOECDM = 0.859 × NOECCD + 0.313	0.70, 1.02	−0.21, 0.84	0.84	0.87
		EnviroTox w. reps	76	11	NOECDM = 0.927 × NOECCD + 0.021	0.74, 1.11	−0.50, 0.54	0.97	0.18

DM: Daphnia magna

DP: Daphnia pulex

CD: Ceriodaphnia dubia

D. magna and C. dubia chronic toxicity data was less homogeneous. The majority of hazard values were reported as NOECs (84%), however, EC10, EC20, and MATC values were also included. All chronic D. magna studies were 21d in duration. The majority of C. dubia chronic studies were 7d in duration (89%), but 8 d duration studies were also included in this analysis in order to expand the chemical comparison between species.

Statistical Analysis

A series of pairwise comparisons were made to describe the correlation between species. For each pairwise analysis, the database was subset to include chemicals with data in both species. Many chemicals had multiple experimental results for a given species. To minimize the influence of experimental variability, regressions were performed with geomean experimental values if more than one datapoint was available. D. pulex and C. dubia acute toxicity results were correlated to the standard D. magna species. Chronic toxicity correlations between the 21d D. magna assay and the 7d C. dubia were also derived. The resulting linear relationships were modeled using orthogonal regressions. This statistical method has been previously used to describe the relationship between the standard acute fish toxicity assay and fish embryo toxicity test (Belanger et al., 2013). Ordinary linear regression minimizes the sum of squared vertical distances on the y-axis. When comparing the toxicity of two test organisms, an orthogonal regression is more appropriate as this minimizes the perpendicular distances between the datapoints (Carroll and Ruppert, 1996). This approach accounts for the uncertainty that exists in the experimental data from both test species. For consistency, D. magna was always plotted on the y axes and the x axes was other species of interest (D. pulex, C. dubia).

To determine the influence of study variability, additional regressions were created using only chemicals that had multiple experimental observations. Currently, the WebICE tool (https://www3.epa.gov/webice/) models species correlations using a log-linear least-square regression model. These correlations were recalculated using the orthogonal approach to allow for more accurate comparisons between the highly curated WebICE database and the more chemically diverse, comparatively less curated EnviroTox database.

Regression slope, intercept, 95% confidence intervals, correlation coefficients, and MSE values were computed using R (mcr and pracma packages).

RESULTS and DISCUSSION

Overview of standard acute and chronic methods

Standard invertebrate acute toxicity testing methodology has been created by several jurisdictions and are used in a variety of different applications including chemical and pesticide registrations and whole effluent toxicity tests. A selection of standard acute daphnid toxicity test guidelines were reviewed including internationally recognized guidelines used to assess the hazard of chemicals (OECD 202 (OECD, 2004), US EPA OPPTS method (USEPA 2016b), ECCC method (ECCC, 2000), ISO method (ISO, 2012), ASTM method (ASTM, 2002)) and two guidelines developed to measure whole effluent toxicity (US EPA OW method (USEPA, 2002c), NIWA (NIWA, 1998)). This effort updates and expands the method review previously conducted (Versteeg et al., 1997). Parameters relating to experimental design, biological endpoints, water chemistry considerations and study validity criteria were summarized (Table 2).

Table 2.

Summary of acute toxicity methods for daphnids

	OECD 202	US EPA OPPTS 850.1010	ISO	ASTM E729-96	ECCC EPS 1/RM/11	US EPA OW EPA-821-R-02-012	NIWA
Year	2004	1996	2016	2002	1996	2002	1998
Region	Europe, Global	United States	−	−	Canada	United States	New Zealand
Toxicant	Chemical	Chemical	Chemical	Chemical	Chemical	Whole effluent	Whole effluent
Species	D. magna, other “suitable” Daphnia species (e.g., D. pulex)	D. magna D. pulex	D. magna	D. magna D. pulex D. pulicaria C. dubia	D. magna D. pulex	D. magna D. pulex C. dubia	C. dubia
Duration	48 h	48 h	24, 48 h	48 h	48 h	24, 48, 96 h	48 h
Endpoint	Immobility	Immobility or Mortality	Immobility	Immobility	Immobility or Mortality	Mortality	Immobility or Mortality
Test statistic	EC50	EC50	EC50	EC50	LC50	LC50	EC50
Age of organisms	<24 h old, not first brood progeny	<24 h old	<24 h old, at least third generation	<24 h old	<24 h old	<24 h old	<24 h old
Concentrations	Minimum of 5 conc. and control with a dilution factor no greater than 3.2	Minimum of 5 conc. in a geometric series with a dilution factor between 1.5 and 2.0	Range of conc. resulting in at least three partial immobilization between 10% and 90%	Minimum of 5 conc. in a geometric series with a dilution factor of 0.6	Minimum of 5 conc. and control	5 conc. and control with ≥ 0.5 dilution series	Minimum of 5 conc. and control
Medium	Surface, well, reconstituted, or dechlorinated tap water	Surface, ground, reconstituted, or dechlorinated tap water	Reconstituted (ISO medium)	Natural, reconstituted, or dechlorinated drinking water	Reconstituted, ground, surface, or dechlorinated municipal water	Receiving, ground, or synthetic water	20% Freshwater, 80% deionized water + stock nutrient solutions
Hardness	>140 mg/L (as CaCO3)	<250 mg/L (preferably <180 mg/L); 40–50 mg/L for testing with metals	250 ± 25 mg/L (as CaCO3)	Not Specified	D. magna : >80 mg/L D. pulex: any hardness	D. magna:160 −180 mg/L CaCO3 D. pulex: 80–90 mg/L CaCO3	Not Specified
Replicates	4 reps, 5 organisms each	2 replicates, 10 organisms each	Not Specified (at least 20 organisms)	At least 2 replicates, 10 organisms each	≥ 10 organisms per concentration	4 replicates, 5 organisms each	3 to 5 replicates, 10 organisms each
Volume	≥ 2 mL per organism	≤ 40 daphnids per liter	≥ 2 mL per organism	Not Specified	≤ 1 daphnid per 15 mL	Not Specified	10 neonates per 15 mL
Temperature	18–22 ± 1 C	20 ± 2 C	20 ± 2 C	Daphnia: 20 C Ceriodaphnia: 25 C	20 ± 2 C	Daphnia: 20 ± 1 C Ceriodaphnia: 25 ± 1 C	25 ± 1 C
Validity criteria	< 10% control mortality; DO ≥ 3 mg/L at end of study	< 10% control mortality; constant conditions must be maintained throughout test; DO must be maintained at >60% saturation	< 10% control mortality; DO ≥ 2 mg/L at end of study	< 10% control mortality; DO must be maintained at > 60% saturation in a renewal or flow-through test, static test at least 40% saturation at end of test	< 10% control mortality	≤ 10% control mortality	≤ 20% control mortality

Daphnid acute toxicity guidelines vary in the diversity of accepted test species. The OECD 202 guideline states “Daphnia magna Straus is the preferred test species although other suitable Daphnia species can be used in this test (e.g., Daphnia pulex)” (OECD, 2004). The ECCC and US EPA OPPTS guidelines are focused on D. magna and D. pulex species. ASTM and US EPA OW guidelines include the option to also test C. dubia.

The goal of each method is to identify the concentration of chemical (or effluent) that results in 50% immobilization (a proxy for mortality). All tests are initiated with <24h old neonates and are run for 48h, though 24h and 96h endpoints are allowed under some guidelines. In practice, it is rare of see acute daphnid toxicity data that deviates from a 48h duration.

Experimental designs are very consistent between acute test methods. A minimum of 20 organisms are used per test concentration, however guidelines differ on how these organisms are split into replicates (e.g., 4 replicates of 5 organisms or 2 replications of 10 organisms). Methods all required a minimum of 5 test concentrations and a control. Suggested dilution factors range between 0.5 and 3.2. Test validity criteria are also very consistent between guideline methods. Control morality must be <10% (or <20% for the NIWA whole effluent guideline). Most methods also include a dissolved oxygen criterion.

Some methods provide refined test conditions suited for D. pulex or C. dubia. Very minor deviations from the standard protocols are described. D. magna are generally cultured in moderately hard or hard water conditions (120–180 mg/L CaCO3), while D. pulex is capable of surviving under softer water conditions. US EPA OW and ECCC both outline hardness considerations for using D. pulex instead of D. magna. Running an acute toxicity test with C. dubia requires a separate experimental deviation. C. dubia are typically cultured at a higher temperature than D. magna and D. pulex (Table 1). All guidelines that accept C. dubia as a standard organism state these assays should also be conducted at this higher temperature. Overall, the methodological differences between guidelines and between species were very minor.

For this research, aselection of standard chronic daphnid toxicity test guidelines were also reviewed, including chronic C. dubia methods from US EPA OW (USEPA, 2002a), ECCC (ECCC, 2007), and ISO (ISO, 2017) and four chronic daphnid methods (OECD 211 (OECD, 2012), USEPA OPPTS 850.1300 (USEPA 2016a), ASTM E1193-20 (ASTM, 2020), ISO 10706:2000 (ISO, 2000)) (Table 3). Chronic daphnid toxicity test aim to identify a concentration associated with reproductive output effects (e.g., number of live neonates per female, time to first brood). Each test is extended long enough to encompass at least a total of 3 reproductive broods. For D. magna, this results in a 21d assay whereas C. dubia assays are significantly shorter due to the faster maturity rate and shorter generation time (Table 1).

Table 3.

Summary of chronic toxicity methods for daphnids

	OECD 211	USEPA OPPTS 850.1300 EPA 712-C-16-005	ASTM E1193-20	ISO 10706:2000	USEPA OW 1002.0 EPA-821-R-02-013	ECCC EPS 1/RM/21	ISO 20665:2008
Year (original)	2012	2016	2020	2000	2002	2007	2008
Region	Europe, Global	United States	−	−	United States	Canada	−
Toxicant	Chemical	Chemical	Chemical, Whole effluent	Chemical, Whole effluent	Whole effluent	Chemical, Whole effluent	Chemical, Whole effluent
Species	D. magna, other “suitable” Daphnia species provided they meet validity criteria, C. dubia referenced under volume considerations	D. magna D. pulex	D. magna D. pulex	D. magna	C. dubia	C. dubia	C. dubia
Duration	21 days	21 days	D. magna: 21 days D. pulex: A three brood test that terminates based on at least 60 % of the controls having three broods, which occurs after approximately 10–14 days	21 days	8 days or when 60% or more of the surviving control females have produced their third brood	8 days or when 60% or more of the surviving control females have produced their third brood	7 ± 1 day
Endpoint	Mortality, Reproduction	Mortality, Reproduction	Mortality, Reproduction	Reproduction	Mortality, Reproduction	Mortality, Reproduction	Mortality, Reproduction
Test Statistic	ECx, NOEC, LOEC	EC50, NOEC, LOEC		ECx, NOEC, LOEC	LC50, EC50, NOEC, LOEC	LC50, ICx (typically IC20 or IC25)	EC10, EC20 or EC50
Age of organisms	≤ 24 h old, not first brood progeny	≤ 24 h old, not first brood progeny	≤ 24 hours old	≤ 24 h old	≤ 24 h old, and within 8h of the same age	≤ 24 h old, and within 12h of the same age	≤ 24 h old and have been taken from a brood comprising at least eight newly born animals
Concentrations	Minimum of 5 concentrations and control with a dilution factor no greater than 3.2	Minimum of 5 concentrations	Minimum of 5 concentrations and control	Minimum of 5 concentrations and control with a dilution factor no greater than 3.2	Minimum of 5 concentrations	Minimum of 7 concentration	Minimum of 5 concentrations and control with a dilution factor no greater than 3.2
Medium	Fully defined medium	Surface or ground water; reconstituted water, dechlorinated tap water	Not specified but recommends reconstituted water, natural fresh water	M7 and M4 media, ASTM reconstituted hard freshwater, natural water	Uncontaminated receiving water, reconstituted water, uncontaminated natural water	Uncontaminated ground, surface water; dechlorinated municipal water, reconstituted water	ELENDT M4 or moderately hard water is recommended
Hardness	>140 mg/L (as CaCO3)	<250 mg/L (preferably <180 mg/L); 40–50 mg/L for testing with metals	Not Specified	>140 mg/L (as CaCO3)	−	80–100 mg CaCO3/L recommended	90 ± 10 mg/L (as CaCO3)
Replicates	10 replicates, 1 organism each	10 replicates, 1 organism each	10 replicates, 1 organism each	10 replicates, 1 organism each	10 replicates, 1 organism each	10 replicates, 1 organism each	10 replicates, 1 organism each
Volume	50–100 mL (smaller volumes for smaller daphnids, e.g. C. dubia)	Not Specified	40 mL/daphnid (Static renewal) 30 mL/daphnid (Flow-through)	Not Specified	15 mL	15 mL	Not Specified
Temperature	18–22 ± 1 C	20 ± 2 C	20 ± 2 C	18–22 C	25 ± 1 C	25 ± 1 C	25 ± 2 C
Food	Should contain 0.1–0.2 mg organic carbon/Daphnia/d	Same diet as reared with	Not specified	Not Specified	Feed 0.1 mL each of YCT and algal suspension per test chamber	0.1 mL YCT, 0.1 mL algae	fish food with 2 algal species or YCT with 1 algal species
Feeding	Feed daily (preferred), or at least 3 times a week	Daily	Daily (or at least 2 feedings/day if Flow-through)	Not Specified	Daily	Daily	Daily
Validity criteria	In control, CV around mean living offspring produced per parent should be ≤25%; Parent mortality <20% at end of test; Mean number of living offspring produced per animal surviving at the end of the test ≥ 60	Control mortality <20%; Each control must produce an average of ≥60 neonates; No ephippia produced by control animals	Test considered invalid if: Mortality in controls >30%; daphnids >24-hr old; appropriate controls not included; test <21 days; mean number of offspring per parent in controls is ≥ 60 young in 21 days; observation of ephippia; temperature, dissolved oxygen did not meet guideline requirements	≤10 % controls immobilized; Mean number of offspring per parent in controls is ≥ 60%; Presence of living males in controls is < 20 % at the end of the test.	≤20% control mortality; average of 15 or more young per surviving female in the control solutions; 60% of surviving control females must produce three broods	First generation control mortality ≤20%; ≥60% controls must produce 3 broods in 8 days; >15 live young produced per surviving female in control solutions during first three broods; No ephippia observed in any control solutions at any time	The mean mortality rate of the adult females at the end of the test does not exceed 20 %; The proportion of adult males does not exceed 10 %; 60 % or more of the adult females produce three broods by the end of the test; The mean number of offspring born per alive adult female at the end of the test is greater than or equal to 15

The statistical metric used to describe a reproductive effect varied. Some guidelines prefer the derivation of No Observed Effect Concentrations (NOEC) or Lowest Observed Effect Concentrations (LOEC) while others favor concentration-response modeling to calculate an ECx value (typically EC10 or EC20). This mirrors the on-going debate within ecotoxicology between regression and hypothesis based test statistics (Green et al., 2013; Landis and Chapman, 2011). Beasley et al completed a data-driven review of chronic D. magna reproductive assays to evaluate the relationship between NOEC values and EC10 and EC20 values (Beasley et al., 2015). The EC10 value was found to be the most appropriate analog for a daphnia chronic NOEC value.

The experimental designs for the chronic toxicity assays were very similar. All assays used 10 replicates per concentration with 1 organism each. A minimum of 5 test concentration and control were used, except for ECCC guideline which required 7 concentrations. Only the OECD 211 and ISO guidelines had specific requirements for concentration spacing, with dilution factors no greater than 3.2.

Chronic C. dubia methods require smaller experimental volumes (15 mL) than the D. magna methods, presumably due to the organisms’ smaller size. The OECD 211 chronic D. magna test is conducted in volumes of 50–100 mL. The OECD 211 guideline has some ambiguity. The OECD 211 guideline emphasizes only D. magna or “suitable” daphnid species are to be tested. However, the guidance indicates “smaller volumes may be possible especially for smaller daphnids e.g., Ceriodaphnia dubia”. A narrow interpretation of the guideline would consider a 3 brood chronic C. dubia assay to be out of scope because 1) C. dubia are not within the genus Daphnia, and 2) a three brood C. dubia assay would not meet the validity criteria of a mean number of ≥60 living offspring per control animal due to smaller clutch sizes. These critiques are not hypothetical and have been raised in regulatory environmental risk assessment discussions.

All chronic tests require the daily addition of food for the duration of the assay. D. magna and C. dubia have different dietary needs (Table 1). Both chronic C. dubia methods require 0.1 mL of both Yeast-Cerophyll-Trout chow (YCT) and algae suspension per test chamber. Feeding guidance is less descriptive for many D. magna methods. OECD 211 method recommends a mixture of live algal cells containing 0.1–0.2 mg organic carbon per daphnid per day.

Assay validity criteria are based on a combination of adult mortality and reproductive output. Control adult mortality must be less than 20% for all methods except for ISO and ASTM guidelines (<10% and <30, respectively). For D. magna assays, all controls must also have 3 reproductive broods with an average of ≥60 neonates per female over the entire test period. C. dubia assays require that at least 60% of the controls have 3 broods, resulting in an average of >15 neonates per female over the test period. For both test species, controls are monitored for the production of ephippia and/or male neonates. This can be an indication of stress and may invalidate the study.

Comparative sensitivity

Understanding the relative sensitivity of organisms is an important cornerstone of ERAs. Interspecies Correlation Estimation (ICE) models can be used to provide a direct toxicity estimation for an organism that lacks toxicity data by extrapolating from a surrogate species (Raimondo and Barron, 2020). This can be especially useful for estimating the sensitivity of endangered species or estimating the sensitivity of additional species within a given taxa. This conceptual approach can also be used as part of a weight of evidence argument to determine if species are likely to be equisensitive.

Environmental regulations require the submission of environmental hazard data generated following specific standard test methodologies. Under EU-REACH regulation, acute invertebrate toxicity information is generated using the standard OECD 202 test method (OECD, 2004). Data from either D. magna or D. pulex fully meets the regulatory requirement. Therefore, these species are considered equivalent under the regulation. It is therefore important to understand the relative sensitivity of these species and the degree of variability that is inherently accepted under regulatory guidelines. The orthogonal regression of D. magna and D. pulex can be used as benchmark to interpret if C. dubia is equisensitive to D. magna within regulatory interpretations.

The inherent variability between species can be interpreted through the slope, y-intercept, and r-value of an orthogonal regression. If the 95% CI of the slope and y-intercept encompasses the null values of 1 and 0, respectively, this would show no apparent significant differences in species sensitivity.

Acute assay comparison:

D. magna and D. pulex acute toxicity data were obtained from the highly curated WebICE database (Table 4). A total of 21 chemicals were identified including 13 insecticides/herbicides, 5 cyclic hydrocarbons and 3 inorganics. This dataset is dominated by specific acting chemicals with two thirds of the compounds having toxicity values in the µg/L or ng/L range. Some materials had multiple experimental values for a given compound. Only 3 chemicals had multiple D. pulex values. Twelve compounds had multiple values for D. magna. A perfect 1:1 relationship would have an intercept of 0, slope equal to 1, and an r-value of 1. The orthogonal D. magna and D. pulex regression showed a near 1:1 relationship with an intercept of 0.165, slope of 0.967, and r-value of 0.99 (Figure 1A, Table 4). Both the slope and y-intercept 95% confidence intervals encompassed the line of unity.

Figure 1.

Relationship between Daphnia magna and Daphnia pulex acute toxicity. Orthogonal relationships were derived using A) data from the highly curated EPA webICE database, B) experimental results from the EnviroTox database, and C) compounds with multiple experimental values in the EnviroTox database. Individual experimental results are denoted with open gray circles. Filled orange circles indicate the geometric mean. Blue line depicts a 1:1 correlation.

A total of 125 chemicals contained D. magna and D. pulex acute toxicity data within the EnviroTox database. This dataset contained a diverse set of compounds and mechanisms of action. Unlike the WebICE dataset, approximately 50% of these compounds act through a narcosis mode of action (MOA). The EnviroTox database is comparatively less curated than WebICE. This may correspond with greater hazard value variability due to inclusion of studies of varying quality, mix of effects based on nominal and analytical concentrations, or other factors. Visually, this higher level of variability can be observed in Figure 1B. The compounds that had the greatest deviation between D. magna and D. pulex often had single experimental observations for one or both species. Single observations can be difficult to interpret because they do not fully account for assay variability. Individual studies may or may not reflect the central tendency. For this reason, the orthogonal regression was recalculated using only chemicals that had multiple experimental values for both species. This dataset included 50 chemicals and 827 experimental observations (Figure 1C, Table 4). This curation increased the r-value (0.95) and corresponded with a lower mean squared error (MSE; 0.30 compared to 0.72), with only slight changes to the orthogonal regression equation. D. pulex and D. magna WebICE and EnviroTox orthogonal regressions demonstrate the equivalency of these species. In all three analysis, the slope and y-intercept 95% CI encompassed or approximated the 1:1 line of unity. Importantly, all 95% CI are calculated based on the plotted geometric means. Given the inherent experimental variability and the uneven replication in the dataset, these confidence intervals may underestimate the variability in regression. The observed D. pulex and D. magna sensitivity deviations are implicitly considered acceptable under a regulatory interpretation, as data from either species fully meets regulatory requirements. We have used the results from these orthogonal regressions to set a data-driven benchmark for what would constitute a suitable D. magna surrogate.

The WebICE dataset used to compared D. magna and C. dubia acute toxicity contained 32 chemicals (258 entries): 9 pesticides/insecticides, 11 organics, and 12 metals/salts/inorganics. The EnviroTox analysis encompasses a different chemical domain. A total of 50 chemicals have multiple experimental acute toxicity tests for both D. magna and C. dubia. Approximately 45% of these compounds are pesticides (insecticides, herbicides). The resulting orthogonal regression had a slope of 0.891 and an intercept of 0.688 (Figure 2B, Table 4). A previous analysis by Versteeg et al compared the acute sensitivity of D. magna, D. pulex, and Simocephalus vetulus to C. dubia (Versteeg et al., 1997). This analysis included 48 materials (organic, inorganic, and effluents). The regression had a very strong correlation (r²=0.96). The authors derived a slope of 0.97; closely aligning with the observations of our own work. Orthogonal regressions completed with the highly curated WebICE dataset and more chemically diverse Envirotox dataset contained overlapping slope and y-intercept 95% confidence intervals. The slope of the C. dubia and D. magna acute regression was ∼1. The y-intercept suggests that C. dubia is either equivalent or slightly more sensitive than D. magna.

Figure 2.

Relationship between Daphnia magna and Ceriodaphnia dubia acute toxicity. Orthogonal relationships were derived using A) data from the highly curated EPA webICE database and B) compounds with multiple experimental values in the EnviroTox database. Individual experimental results are denoted with open gray circles. Filled orange circles indicate the geometric mean. Blue line depicts a 1:1 correlation.

Collectively, the orthogonal regressions between D. magna, D. pulex, and C. dubia can be examined to determine if these species are acutely equisensitive. In all cases, these species had a near perfect 1:1 relationship with a slope of ∼1. Regressions created using highly curated Web-ICE data fully encompassed the graphical origin. Though the y-intercept of the EnviroTox D. magna and C. dubia regressions did not encompass zero, this deviation was equivalent to the y-intercept from the D. magna and D. pulex regressions. We can therefore conclude that this small level of variation is acceptable under regulatory guidelines, and all three species are acutely equisensitive.

MOA comparison:

The influence of MOA on relative D. magna and C. dubia sensitivity was further explored. A Consensus MOA scheme has been recently developed that allows for a transparent assignment of narcotic, specific acting, or unclassified MOAs based on consensus MOA assignments from 4 independent models (Verhaar, ASTER, TEST, OASIS) (Kienzler et al., 2019). Consensus MOA assignments are available in the EnviroTox database and were leveraged for this analysis. The 1:1 relationship between D. magna and C. dubia was consistent for all consensus MOAs (Table 5). MOA categories have historically been created based on fish toxicological responses and may not be fully descriptive of invertebrate MOA. It is therefore unsurprising that some pesticides are classified as narcotics or unclassified under the Consensus MOA scheme.

Table 5.

Daphnia magna and Ceriodaphnia dubia orthogonal regressions based on mode of action. All hazard units are denoted in µg/L units.

Comparison	Test Type	Consensus MOA	# Entries	# Chems	Equation	Slope 95% CI	Intercept 95% CI	r	MSE
D. magna, C. dubia	Acute	Narcosis	354	21	LC50DM = 0.910 × LC50CD + 0.205	0.76, 1.05	-0.06, 0.46	0.95	0.21
		Specific Acting	259	14	LC50DM = 1.054 × LC50CD + 0.907	0.84, 1.27	0.23, 1.57	0.95	0.17
		Unclassified	259	15	LC50DM = 0.816 × LC50CD + 0.572	0.50, 1.13	0.03, 1.12	0.84	0.36

DM: Daphnia magna

CD: Ceriodaphnia dubia

Experimental variability:

Many compounds had a high degree of replication in both the WebICE and EnviroTox database. This allows for an exploration of study variability. Some compounds had relatively low coefficients of variation (CV). The standard reference toxicant sodium chloride had 16 C. dubia acute toxicity results in the WebICE database. LC50 values ranged from 1.42 to 3.03 g/L (average=2.16 g/L; CV=20.8%). This low CV speaks to the assay precision and accuracy for this specific compound. Six D. magna malathion acute toxicity tests are contained within WebICE. Hazard values ranged from 1.6 to 2.2 µg/L (average 1.83 µg/L, CV=14%). For malathion, a higher degree variation was observed with C. dubia. A total of 7 acute toxicity tests were available ranging from 0.58 to 2.19 µg/L (average 1.53 µg/L, CV=41%). It is unclear if the difference in CV was due to do something unique about the species/assay, study quality, or other factors.

High CV values were not uncommon in this dataset, nor were they isolated to one cladoceran species. Endrin D. magna acute toxicity values had a very wide range (4.2–320 µg/L). An average effect concentration of 86 µg/L was calculated, corresponding to a CV of 98%. Significant amount of variability was especially apparent with metals. This is despite the careful manual data curation and normalization procedures employed in the WebICE database. A total of 21 C. dubia and 8 D. magna silver acute toxicity experiments were available in this database. Both species had toxicity values spanning over 3 orders of magnitude. D. magna and C. dubia had CVs of 85% and 179%, respectively.

Several factors can be associated with assay variability including technical expertise of the researcher, small variations in water quality parameters and test conditions, organism age and condition, quality (or type) of food provided, solution preparation procedures, solution stability, and the endpoint being considered. The precision of the D. magna, D. pulex, and C. dubia acute toxicity assays, as defined by percent coefficient of variation, has been explored. Interlaboratory examinations have routinely showed CV ranging between 14–80% for standard reference toxicants for all three species (USEPA, 2002c). CVs from the broader literature regarding repeatability of chronic toxicity tests are less abundant than for acute toxicity tests but some comparisons exist. Moore et al. (2000) showed intra- and interlaboratory variability for C. dubia reference toxicant tests with NaCl ranged from 15.7–55.7% and 13.4–17.3%, respectively. Similar values can be found in the aquatic toxicity literature (Busquet et al., 2014).

Chronic assay comparison:

D. magna and C. dubia chronic toxicity assays both extend for a total of 3 reproductive broods. Due to differences in their maturity rates and generation times, C. dubia chronic assays can be completed in only ∼7d while D. magna assays require 21d to complete (Table 2). There is a concern that this shorter duration (and smaller size) may result in different sensitivity due to the time needed to reach internal equilibrium with the test solution.

Versteeg et al compared sensitivity of “Daphnia” (combining D. magna, D. pulex, D. carinata data) to C. dubia (Versteeg et al., 1997). A total of 16 chemicals (12 organic, 4 inorganic) and 4 effluents were used in this analysis. No statistically significant difference in species sensitivity was observed across all compounds. All NOEC values were within an order of magnitude for each species with the exception of trichloromethane, where C. dubia was two orders of magnitude more sensitive than D. magna. The linear regression showed a significant linear relationship (r²=0.62) and a slope of ∼0.90.

A total of 51 chemicals had chronic D. magna and C. dubia data in the EnviroTox database (Table 4). Many chemicals had multiple experimental values. As before, the chronic compounds that had pronounced differences between the two species often had very limited experimental replication (Figure 3A). Three of the compounds with the largest differences in species sensitivity were endosulfan, carbofuran, and diclofenac (D. magna/C. dubia ratio= 17, 27, 18, respectively). These compounds had a single experimental value for one or both species. Endosulfan and diclofenac both have high logP values (3.83 and 4.51, respectively). Ten other compounds had logP>3. However, the hazard values of these compounds varied less than an order of magnitude between species and often within a factor of 2. There was no apparent difference in species sensitivity based on logP.

Figure 3.

Relationship between Daphnia magna and Ceriodaphnia dubia chronic toxicity. Orthogonal relationships were derived using A) experimental results from the EnviroTox database and B) compounds with multiple experimental values. Individual experimental results are denoted with open gray circles. Filled orange circles indicate the geometric mean. Blue line depicts a hypothetical 1:1 correlation.

When the EnviroTox dataset was restricted to only chemicals with multiple experimental results for both species, 11 chemicals (76 entries) remained. The resulting orthogonal regression demonstrated a near 1:1 relationship between chronic D. magna and C. dubia assays. The regression had a y-intercept of 0.021 and an r-value of 0.97. The slope of 0.927 supports the results of Versteeg et al (linear regression slope = 0.90)(Versteeg et al., 1997). The orthogonal regression using all data and only replicate chronic data did not substantially differ. Both regressions had slope and y-intercept 95% confidence intervals that fully encompassed the line of unity (slope of 1, y-intercept of 0).

CONCLUSIONS

Filter feeding, pelagic freshwater species remain the most common (and usually only) invertebrate species tested for standard environmental risk assessments. Acute and chronic toxicity testing is typically done with one species, Daphnia magna. Standard regulatory test guidelines allow for data generation for several different cladocera that are closely related (e.g., D. pulex, and sometimes C. dubia). Both D. magna and C. dubia are routinely cultured and used globally in standard toxicity tests. Additionally, these species fulfill similar ecological niches and have global geographic distributions. Under strict interpretations of OECD test guidelines, C. dubia are not considered as a fully accepted test species for chronic reproductive assays (OECD 211). The 7d chronic C. dubia assays has significant advantages over 21d chronic D. magna assay – most notably the reduced experimental duration and smaller exposure volumes. Based on analyses of a chemically-limited but highly curated database (Web-ICE) and the chemically-diverse but less well curated database (EnviroTox), there was no evidence of significant differences in acute or chronic sensitivity of D. magna and C. dubia. Further, the variability in the acute D. magna and C. dubia regressions were of the same magnitude as D. magna and D. pulex regressions. Slope and y-intercept values were also comparable. Using this data drive benchmark as our point of comparison, we conclude that C. dubia and D. magna are equisensitive in both acute and chronic standard assays.

Data availability statement:

Data, associated metadata, and calculation tools are available from the corresponding author (Connors.ka@pg.com).