Table 2.

In vitro studies of biological effects from DEE after combustion of biodiesel (B100) and biodiesel blends compared to DF.

Authors	Cells/Tissues	Endpoints	Engine	Fuels	Test cycles	Extraction	Biological effects	Remarks
Grägg 1994	S. typhimurium (TA98, TA100) Sub-cellular	Bacterial mutagenicity Ah- receptoraffinity	11 L Scania DSC 1127 truck diesel engine	DF, DF with 2000 ppm ethyl-hexylnitrate, MK1, MK2, RME, B5, B30	City-Line Cycle, Univ. Brunsvig, Germany, ECE R 49	No description of details	RME particle extracts up to sixfold less mutagenic than DF.Blends produced stronger mutagenicity than pure fuels.No coherent results from the Ah-receptor assays.	Results not sufficiently documented. Ah-receptor-assay of questionable value concerning DEE
Carraro et al. 1997	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	1.9L DI and IDI turbo diesel, EGR	DF with 1000 ppm sulfur, RME	European standard test cycle (ECE-EUDC)	Soxhlet with benzene and acetone	RME up to two-fold less mutagenic than DF, good correlation with PAH and nPAH analyses.	Methods and results of chemical analysis not sufficiently documented.
Bagley et al. 1998	S. typhimurium (TA98, TA98NR, TA98/1.8–DNP6)	Bacterial mutagenicity	1983 7L Caterpillar 3304 PCNA Diesel engine± DOC	DF, SME	light- and heavyduty transient test cycles of the U.S. Bureau of Mines (USBM)	Soxhlet with DCM, 24h in the dark	Mutagenicity of particle extracts stronger compared to condensates. SME with DOC up to four-fold less mutagenic than DF. DOC reduced mutagenicity of SME and DF to a comparable extent.	PM and PAH with SME reduced, SOF slightly increased.
Bünger et al. 1998	S. typhimurium (TA97a, TA98, TA100, TA102) Mice fibroblasts (L929)	Bacterial mutagenicity Cytotoxicity	1.9 L DI VW turbo diesel engine, EGR, DOC	DF, RME	US- FTP-75. European MVEG-A, incl. cold start (MVEG-A1).	Soxhlet with DCM, 24h in the dark	RME up to five-fold less mutagenic than DF, strongest mutagenicity under cold start conditions. RME stronger cytotoxic than DF.	RME led to an increase of soluble PM.
Bünger et al. 2000a	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	One Cylinder test engine Fary-mann 18D	DF, LS-DF, RME, SME	ECE R 49	Soxhlet with DCM, 12h in the dark	Mutagenicity of DF about two-fold stronger compared to SME, LSDF, and RME. Sulfur content and high engine load were associated with strong mutagenicity of PM.	PAH content in DF- and SMEexhaust increased compared to LSDF and RME.
Bünger et al. 2000b	S. typhimurium (TA98, TA100) Mice Fibroblasts (L929)	Bacterial mutagenicity Cytotoxicity	4 Cylinder Fendt 306 LSA tractor-engine	DF, RME	ECE R 49	Soxhlet with DCM, 12 h in the dark	DF at heavy load about four-fold stronger mutagenic. RME at partial load about four-fold stronger cytotoxic.	RME: TPM↑ SOF↓ DF, at partial load: trend towards ultrafine particles ↑
Morin et al. 2000	Rat lung slices	Apoptosis and biochemical markers of inflammation	1 Cylinder diesel test engine, 230 ccm	DF, RME, and B30	Engine operation at 3000 min-1	No extraction, direct exposure to 5, 10, 15, 25, 60, and 85% filtered and unfiltered exhaust for 3 hours	No clear trend for TNFα.GSHdepletion: up to 70% with un-filtered and 30% with filtered exhaust. DF led to apoptosis, RME and the blend did not.	Inconclusive results. The impact of apoptosis concerning diesel exhaust is not clear; it may be beneficial in case of genotoxic effects.
Kado et al. 2001	S. typhimurium (TA98)	Bacterial mutagenicity	5.9 L Cummins ISB turbo diesel, ± DOC	DF, B20, B50, B100 from Rapeseed ethyl ester (REE),	US Code of Fed Reg. 40, Part 86, cold- and hot-start	Sonication with dichloromethane (DCM)	B100 (REE) much less mutagenic than DF. B20 had strongest mutagenicity. B20 and B50 with DOC up to 10-fold stronger mutagenic than without DOC.	Results for increased bacterial mutagenicity with DOC for B20 and B50 is not discussed in the paper
Kado and Kuzmicky 2003	S. typhimurium (TA98)	Bacterial mutagenicity	6 Cylinder. DI 11.1 L Detroit turbo-diesel	DF, CME, SME, PLME, YGME, and BTME	US Code of Federal Regulations 40. Part 86. Subpart N	DCM (no details given)	PM and mutagenicity at cold- stronger compared to hot start. Reverse mutations /KWh: DF > CME > SME = PLME = YGME > BTME.	Biofuels from animal fat showed strong direct mutagenicity.
Bünger et al. 2004	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	VW 1.9 L-TDI, ± DPF and during regeneration	DF, RME		Soxhlet with DCM, 12h in the dark	With DPF: mutagenicity↓ Regeneration: mutagenicity↑, up to 2.5-fold stronger with the aged catalyst, most pronounced for DF.	PM was reduced by DPF and increased during regeneration phase of the DPF.
Turrio-Baldassarri et al. 2004	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	IVECO 8360. 46R, 7.8 L turbodiesel, Euro II	DF, B20	ECE R 49	ASEextractorfilter: toluene, PUF: 1/1n-hexane/acetone	Pure fuels and blends did not differ concerning mutagenicity.	Strong variability of the Ames-Test results. Extraction at high temperature and pressure.
Bünger et al. 2006	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	One Cylinder test engine Fary-mann K54, ± DOC	DF, LSDF, RME, SME	ECE R 49	Soxhlet with DCM, 12h in the dark	With DOC mutagenicity up to 20%↓ At heavy load about 70% ↑ in case of RME and SME, at idling heterogeneous results.	Stronger mutagenicity with DOC under certain conditions; possibly correlated with NOx.
Krahl et al. 2006	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	5.9 L IVECO Turbodiesel tector F4A with SCR-System	DF, RME, RME with content of 10 ppm phosphor (RME10)	E46 Endurance test and European standard cycle (ESC)	Soxhlet with DCM, 12h in the dark	No significant mutagenicity was detected with the brandnew SCR. Slight mutagenic effects were observed after an engine operation of 1000 hours. No influence of phosphor content on mutagenicity.	SCR seems to be a very effective exhaust after-treatment. Good durability of the SCR towards phosphor.
Ackland et al. 2007	Human alveolar cells (A549)	Apoptosis	1.6L VW-engine	DF, RME, and blends thereof	ECE Euro 2	Three days in DCM (no further details)	Induction of apoptosis was stronger for DF than for RME, strongest effect observed for B20.	The impact of apoptosis concerning adverse health effects of DEE is unclear.
Bünger et al. 2007	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	Mercedes OM 906 LA 6.4 L turbo-diesel, Euro III	DF, RME, Rapeseed vegetable oil (RVO)	Stationary European cycle (ESC)	Soxhlet with DCM, 12 h in the dark	Weak mutagenicity of PM extracts and condensates from DF and RME, 9.7– up to 59-fold stronger mutagenicity in TA98, 5.4– up to 22.3-fold in TA100, condensates of the RVO fuels caused an up to factor 13.5 stronger mutagenicity	RVO was combusted with and without preheating to 70°C (two-tanktechnology). The engine itself was not adapted for RVO combustion.
Krahl et al. 2008	S. typhimurium (TA98, TA100)	Bacterial mutagenicity	Mercedes OM 906 LAMAN D08 36 LFL51 turbo-diesel, 6.8 L, DPF, Euro IVAVL502.019, 1.5 L	DF, B5, B10, B20, B30, B40, B50, B100 from RME	ESCEuropean Transient Cycle (ETC)Rated power	Soxhlet with DCM, 12 h in the dark	Blends showed increased mutagenicity with three enginesStrongest effect was observed for B20 (up to three-fold compared to the pure fuels): B20 > B10 > B50 = B5 > DF > RME.
Liu et al. 2008	Vibrio fischeri, BEAS-2B cells (human bron-chial cell-line)	Microtox-test MTT- assay	Dieselgenerator, 13 kW	DF, B10, B30, B50, B75, B100 from PME	constant load	Soxhlet with DCM and n-hexane	PME yielded increased toxicity, most pronounced for B50.	PME caused increased PM emissions.
Jalava et al. 2010	Mouse RAW 264.7 macrophages	COMET Assay, apoptosis, cytotoxicity, ROS, TNF-α	1.1 L IDI Kubota D1105-T Diesel, EURO II	DF, RME, HVO	ISO standard steady state cycle (8178–4:1996)	Sonication for 2 × 30 min. with methanol	Concentrationrelated DNA strand breaks and toxicity of the extracts, no differences regarding DF, RME, HVO, and use of DOC. DF and HVO stronger TNF-α induction, ROS induction by HVO and RME.	Detailed information about the used catalyst is lacking. All effects were referred to TPM and not to KW/h or exhaust volume.