Thiophanate-methyl

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-06-22 to 1989-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
- Sample storage conditions before analysis: Test samples were injected directly.

Vehicle:

yes

Remarks:

1 % Tween 80-dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Fresh stock solutions of the test item dissolved in 1 % Tween 80-dimethylformamide were prepared daily for dispensing by the continuous flow apparatus. The solvent stock solutions for the test series were 0.10, 0.32, 1.0, 3.2 and 10 mg/mL.

Test organisms (species):

Salmo sp.

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: Salmo gairdneri
- Source: Belleau Bridge Trout Farm, Lincolnshire, U.K.
- Length at study initiation (length definition, mean, range and SD): 4.4 cm (SD = 0.1 cm)
- Weight at study initiation (mean and range, SD): 2.13 g (SD = 0.18 g)
- Feeding during test: yes
- Food type: Commercial trout pellets daily

ACCLIMATION
- Acclimation period: 7 days prior to test
- Health during acclimation: Approximately 1 % mortality in 7 days prior to the test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

50 mg/L as CaCO3

Test temperature:

14 ± 1 °C

pH:

7.7 - 7.9

Dissolved oxygen:

> 9.6 mg O2/L

Nominal and measured concentrations:

Nominal Concentration: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L; The mean percentage of the nominal concentrations obtained from the entire study were near nominal and it is considered, therefore, that the test results should only be determined on the basis of the nominal values and not the measured values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Size of vessel: 20 L
- Material, size, headspace, fill volume: Glass, 20 L
- Aeration: None, the diluent supply only was aerated
- Type of flow-through: peristaltic diluter
- Renewal rate of test solution (frequency/flow rate): Continuous at 59 mL/min
- No. of organisms per vessel: 10 animals per test concentration
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 1.07 g bodyweight/L (static volume), 0.25 g body-weight/L (volume in 24 hours) and 4.25 tank volumes per day.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by the addition of sodium thiosulphate.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED: Absence of respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Marked reactions to exposure (other than death) were lethargy and loss of equilibrium. Statistical analysis of the data shows that there were no significant dose related effects on growth in terms of both length and weight (P > 0.05) of the controls and all surviving test groups.

The mean percentage of the nominal concentrations obtained from the entire study were near nominal and it is considered, therefore, that the test results should only be determined on the basis of the nominal values and not the measured values.

Table 1: Analytical Measurements

Nominal Concentration (mg/L)	Mean percentage of nominal concentration
Control	-
0.010	136
0.032	129
0.10	117
0.32	94
1.00	95

 

Table 2: Analytical RESULTS

Sample	Nominal Concentration (mg/L)	Concentration found (mg/L)	Concentration expressed as % of nominal
Day 0	Control	*	*
	0.010	*	*
	0.032	*	*
	0.10	*	*
	0.32	*	*
	1.0	*	*
Day 4	Control	*	*
	0.010	*	*
	0.032	*	*
	0.10	0.0949	94.9
	0.32	0.295	92.2
	1.0	0.915	91.5
Day 5	Control	+
	0.010	0.0178	178.0
	0.032	0.0603	188.4
	0.10	0.222	222.0
	0.32	0.250	78.1
	1.0	0.937	93.7
Day 6	Control	0.00469	-
	0.010	0.0118	118.0
	0.032	0.0688	215.0
	0.10	0.218	218.0
	0.32	0.206	64.4
	1.0	0.753	75.3
Day 7	Control	0.00632	-
	0.010	0.0313	313.0
	0.032	0.0319	99.7
	0.10	0.128	128.0
	0.32	0.313	97.8
	1.0	1.01	101.0
Day 8	Control	0.00353	-
	0.010	0.00980	98.0
	0.032	0.0425	132.8
	0.10	0.114	114.0
	0.32	0.365	114.1
	1.0	1.06	106.0
Day 11	Control	0.00941	-
	0.010	0.0118	118.0
	0.032	0.0372	116.3
	0.10	0.115	115.0
	0.32	0.264	82.5
	1.0	0.942	94.2
Day 13	Control	None detected	-
	0.010	0.00717	71.7
	0.032	0.0407	127.2
	0.10	0.0934	93.4
	0.32	0.335	104.7
	1.0	0.848	84.8
Day 15	Control	0.00445	-
	0.010	0.0103	103.0
	0.032	0.0418	130.6
	0.10	0.0817	81.7
	0.32	0.290	90.6
	1.0	0.893	89.3
Day 18	Control	None detected	-
	0.010	0.00821	82.1
	0.032	0.0220	68.8
	0.10	0.101	101.0
	0.32	0.308	96.3
	1.0	0.945	94.5
Day 20	Control	0.0108	-
	0.010	0.0112	112.0
	0.032	0.0348	108.8
	0.10	0.122	122.0
	0.32	0.285	89.1
	1.0	1.07	107.0
Day 22	Control	Interference	-
	0.010	Interference	_
	0.032	Interference	-
	0.10	0.0630	63.0
	0.32	0.307	95.9
	1.0	1.11	111.0
Day 25	Control	0.000726	-
	0.010	0.0145	145.0
	0.032	0.0247	77.2
	0.10	0.0693	69.3
	0.32	0.329	102.8
	1.0	0.757	75.7
Day 28	Control	None detected	None detected
	0.010	0.0153	153.0
	0.032	0.0484	151.3
	0.10	0.0994	99.4
	0.32	0.341	106.6
	1.0	1.17	117.0

* A method of analysis was developed prior to the study, but unfortunately when the Day 0 samples were analysed there was unanticipated chromatographic interference. This was not overcome until Day 4 of the study.

+ Lost due to spillage.

Validity criteria fulfilled:

yes

Conclusions:

In a prolonged toxicity (28-days) of the test item to Rainbow trout (Salmo gairdneri) according to OECD 204, the LC50 was determined to be 0.60 mg/L and the NOEC was determined to be 0.32 mg/L.

Executive summary:

In a prolonged toxicity (28-days) of the test item to Rainbow trout (Salmo gairdneri) according to OECD 204 in a flow through test system. Fresh stock solutions of the test item dissolved in 1 % Tween 80-dimethylformamide were prepared daily for dispensing by the continuous flow apparatus. The test concentrations were 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. To ensure the stability of the test concentrations, continuous renewal of test media and verification by chemical analysis on Days 0, 4, 6, 8, 11, 13, 15, 18, 20, 22, 25 and 28 was carried out. Additional samples were taken on days 5 and 7. The criterion for death was determined by the absence of respiratory movement and response to physical stimulation. The LC₅₀ was determined to be 0.60 mg/L and the NOEC was determined to be 0.32 mg/L.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-08-07 to 2014-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

Adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
Aliquots of exact 25 µL of both IS solutions (thiophanate-ethyl and carbendazim-D4) were pipetted one by one into 1.8 mL HPLC vials; afterwards analytical sub-samples of 1.5 mL of the pre-diluted water samples were added. After tightly closing and manual shaking aliquots of 1.0 µL (analyte 1, TPM) or 10.0 µL (analyte 2, MBC) of the mixtures were analysed successive by LC-MS/MS using separate methods for the two analytes. Where necessary, sample aliquots less than 1500 µL were diluted with a mixture of acetonitrile/'holding- and dilution-water'/formic acid (500/500/1, v/v/v) in a pre-dilution step.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Nominal initial test concentrations were 0.125, 0.25, 0.5, 1 and 2 mg test item. Four replicate vessels per test concentrations were installed. The stock solutions for nominal concentrations of 0.125, 0.25 and 0.5 mg/L were prepared by solving 184 mg of the test item with 5000 µL HCl (37 %) in 10 L dilution water under stirring at room temperature for 24 h. HCl was added to the stock solution to prevent formation of carbendazim being the major metabolite of the test item. After 24 h the stock solution was mixed with another 30 L of dilution water. The stock solutions for nominal concentrations of 1000 and 2000 µg/L were prepared by solving 4 times 276 mg of the test item with 2500 µL HCl (37 %) in 20 L dilution water under stirring at room temperature for 24 h. The final test media were prepared using the flow through system, by diluting application solutions with dilution water in the mixing vessels.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Fraunhofer Institute, Schmallenberg, Germany.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Eggs were collected in a glass spawning-tray which was placed at the bottom of the holding vessels
- Subsequent handling of eggs: The tray was covered with a stainless steel lattice to prevent adult fish from eating eggs.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Test temperature:

24.6 – 25.7 °C

pH:

7.9 - 8.4

Dissolved oxygen:

93 – 103 µg/L

Nominal and measured concentrations:

Nominal: 125, 250, 500, 1000 and 2000 µg/L
Measured conc: 123, 256, 509, 939 and 1870 µg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups: glass spawnin-tray
- Test vessel: full glass aquaria
- Material, size, fill volume: glass, 28 L and 24 – 25 L
- No. of fertilized eggs/embryos per vessel: 60
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water was used according to the OECD-Guideline 210

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 12 h/12 h
- Light intensity: 1000 lux

EFFECT PARAMETERS MEASURED: Qualitative observations on hatching and survival were made daily.

VEHICLE CONTROL PERFORMED: dilution water (without addition of test item) was used as a control

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Remarks on result:

other: CL: 0.147 - 0.645 mg/L

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

0.509 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.87 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.87 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.87 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

No adverse effect could be observed on the fish larvae concerning the endpoint hatching success. Post hatch survival was inhibited by the test item at mean measured concentrations of > 0.509 mg/L and > 0.256 mg/L after 14 and 21 days, respectively. However, no statistically significant differences to the control were found at these test levels at all later observation dates. After 28 and 35 days the post hatch survival was affected at 0.939 mg/L and 1.870 mg/L.

Table 1: Measured concentrations of the test item and the residual carbendazim

Nominal concentration			Mean measured concentration
thiophanate-methyl	Carbendazim		thiophanate-methyl		Carbendazim
[µg/L]	[µg/L]	Replicate	[µg/L]	[µg/L]	[µg/L]	[µg/L]
Control	Control	A	< LOQ	-	< LOQ	-
		B	< LOQ	-	< LOQ	-
		C	< LOQ	-	< LOQ	-
		D	< LOQ	-	< LOQ	-
125	70	A	131	105	14.4	20.7
		B	130	104	14.4	20.6
		C	115	91.7	15.1	21.7
		D	115	92.1	14.6	20.9
		Mean	123	98.1	14.6	21.0
		SD	9.1	7.2	0.3	0.5
250	140	A	262	105	20.3	14.6
		B	262	105	20.5	14.7
		C	252	101	21.7	15.5
		D	250	100	21.0	15.1
		Mean	256	103	20.9	15.0
		SD	6.4	2.6	0.6	0.4
500	279	A	475	94.9	25.3	9.1
		B	477	95.3	25.2	9.0
		C	545	109	31.5	11.3
		D	540	108	30.2	10.8
		Mean	509	102	28.1	10.0
		SD	38.9	7.8	3.3	1.2
1000	558	A	905	90.5	41.9	7.5
		B	897	89.7	42.3	7.6
		C	970	97.0	47.1	8.4
		D	985	98.5	45.3	7.1
		Mean	939	93.9	44.2	7.9
		SD	44.5	4.4	2.5	0.4
2000	1117	A	1912	95.6	73.0	6.5
		B	1887	94.3	74.0	6.6
		C	1835	91.8	66.7	6.0
		D	1845	92.2	65.9	5.9
		Mean	1870	93.5	69.9	6.3
		SD	36.0	1.8	4.2	0.4

LOQ thiophanate-methyl = 50 µg/L. LOQ carbendazim = 1 µg/L.

 

Table 2: Early Life stage: Hatch, survival and growth.

	Nominal concentration of the test substance [µg a.s./L]
	Control	125	250	500	1000	2000
	Mean measured concentration [µg a.s./L]
	Control	123	256	509	939	1870
Number of introduced eggs [n]	60	60	60	60	60	60
Hatching day 3 pf [%]	26.7 ± 9.4	28.3 ± 6.4	28.3 ± 14.8	33.3 ± 14.4	26.7 ± 21.1	16.7 ± 3.8
Hatching day 4 pf [%]	90.0 ± 8.6	83.3 ± 15.9	86.7 ± 5.4	76.7 ± 8.6	70.0 ± 12.8	78.3 ± 17.5
Hatching day 5 pf [%]	98.3 ± 3.3	93.3 ± 5.4	98.3 ± 3.3	95.0 ± 6.4	91.7 ± 12.6	96.7 ± 6.7
Hatching day 6 pf [%]	100 ± 0.0	100 ± 0.0	95.0 ± 10.0	100 ± 0.0	98.3 ± 3.3	100 ± 0.0
Hatching day 7 pf [%]	100 ± 0.0	100 ± 0.0	100 ± 0.0	100 ± 0.0	100 ± 0.0	100 ± 0.0
Post hatch survival day 14 pf [%]	95.0 ± 3.3	90.0 ± 6.7	88.3 ± 6.4	85.0 ± 6.4*)	75.0 ± 11.3*)	70.0 ± 3.8*)
Post hatch survival day 21 pf [%]	91.7 ± 6.4	85.0 ± 6.4	83.3 ± 8.6*)	83.3 ± 6.7*)	75.0 ± 11.3*)	68.3 ± 6.4*)
Post hatch survival day 28 pf [%]	90.0 ± 8.6	85.0 ± 6.4	83.3 ± 8.6	81.7 ± 8.4	75.0 ± 11.3*)	68.3 ± 6.4*)
Post hatch survival day 35 pf [%]	90.0 ± 8.6	85.0 ± 6.4	83.3 ± 8.6	81.7 ± 8.4	73.3 ± 12.4*)	66.7 ± 5.4*)
Mean length, day 35 pf [cm]	1.40 ± 0.04	1.54 ± 0.05	1.58 ± 0.06	1.57 ± 0.09	1.55 ± 0.09	1.52 ± 0.09
Mean group dry weight [mg]	68.2 ± 9.5	108.5 ± 21.9	126.1 ± 16.2	119.4 ± 30.4	103.5 ± 21.0	92.4 ± 17.5
Mean single dry weight [mg]	5.0 ± 0.3	8.5 ± 1.4	10.1 ± 0.7	9.8 ± 2.5	9.6 ± 2.4	9.2 ± 1.6

Mean values ± standard deviation are presented. All treatment data was evaluated against the control.

*) statistical significant inhibited compared to control, Williams test, p<0.05, one-sided smaller

 

Table 3: Analytical and statistical results of the fortification experiments for TPM.

Fortification level (F)		Analytical results			Statistical results
No. (label) and level	Analyte conc.	Dilution factor	LC-MS/MS quan. data	Measured analyte conc.	Recovery	Mean recovery	Standard deviation SD	Relative standard deviation, RSD
-	[µg/L]	-	[µg/L]	[µg/L]	[%]	[%]	[%]	[%]
F0a - F0b, Blanks	-	1	n.d. *)	< LOQ	-	-	-	-
		1	n.d. *)	< LOQ	-
F1a - F1e, LOQ level	50.0	1	46.189	46.2	92.4	86.5	3.63	4.14
		1	42.353	42.4	84.7
		1	43.683	43.7	87.4
		1	41.506	41.5	83.0
		1	42.550	42.6	85.1
F2a - F2e, 10 X LOQ level	500	1	507.565	508	101.5	102.3	0.83	0.81
		1	517.529	518	103.5
		1	508.120	508	101.6
		1	510.671	511	102.1
		1	513.549	514	102.7
F3a - F3e, Residue level	2000	1	1945.955	1946	97.3	98.7	1.32	1.34
		1	1986.852	1987	99.3
		1	2011.976	2012	100.6
		1	1953.879	1954	97.7
		1	1976.278	1976	98.8
Overall mean (n = 15):						95.6	7.31	7.63

*) n.d. = not detected

Mass transition used for quantification of TPM: m/z 343.15 — m/z 151.15.

 

Table 4: Analytical and statistical results of the fortification experiments for MBC

Fortification level (F)		Analytical results			Statistical results
No. (label) and level	Analyte conc.	Dilution factor	LC-MS/MS quan. data	Measured analyte conc.	Recovery	Mean recovery	Standard deviation SD	Relative standard deviation, RSD
-	[µg/L]	-	[µg/L]	[µg/L]	[%]	[%]	[%]	[%]
F0a - F0b, Blanks	-	1	n.d. *)	< LOQ	-	-	-	-
		1	n.d. *)	< LOQ	-
F1a - F1e, LOQ level	1.0	1	0.999	0.999	99.9	94.9	3.99	4.20
		1	0.974	0.974	97.4
		1	0.954	0.954	95.4
		1	0.907	0.907	90.7
		1	0.911	0.911	91.1
F2a - F2e, 10 X LOQ level	10.0	1	10.048	10.0	100.5	96.1	2.58	2.69
		1	9.561	9.56	95.6
		1	9.376	9.38	93.6
		1	9.565	9.57	95.7
		1	9.483	9.48	94.8
F3a - F3e, Residue level	100	1	101.043	101	101.0	99.4	1.39	1.40
		1	100.255	100	100.3
		1	98.228	98.2	98.2
		1	97.736	97.7	97.7
		1	99.771	99.8	99.8
Overall mean (n = 15)						96.8	3.30	3.41

*) n.d. = not detected

Mass transition used for quantification of MBC: m/z 192.2 — m/z 160.2.

 

Validity of the test

All validity criteria for controls mentioned by the OECD guideline were met:

- the dissolved oxygen concentration was between 60 and 100 % throughout the test;

- the water temperature did not differ by more than ± 1.5 °C between successive days at any time during the test and was kept within 25 °C ± 2 °C

- the survival rate (post hatch) in the controls was greater than 70 %. 

 

Table 5: Results of the Fish early life stage toxicity test on TPM: Effect concentrations

Parameter	NOEC [mg/L]	EC10 [mg/L]	CL [mg/L]
Hatching success	>1.870	-	-
Post hatch survival	0.509	0.390	0.147 – 0.645
Length	>1.870	-	-
Weight	>1.870	-	-

All values are based on mean measured concentrations of thiophanate-methyl (TPM).

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to fish early life stages was determined according to OECD 210. For this purpose, fertilised eggs of zebrafish (Danio rerio) were exposed to five test item concentrations. Hatch, post hatch survival and growth by means of length and weight measurements were recorded. The NOEC based on the mean measured concentrations, was determined to be 0.509 mg/L, for the endpoint post hatch survival. The respective EC10 value was calculated to be 0.393 mg/L (CL: 0.147 – 0.45 mg/L).

Executive summary:

The toxicity of the test item to fish early life stages was determined according to OECD 210. For this purpose, freshly fertilised eggs of zebrafish (Danio rerio) (less than 4 hours of age, post fertilisation) were exposed to 0.125, 0.25, 0.5, 1 and 2 mg/L. A dilution water control was run in parallel. Water samples were analysed using LC-MS at an LOQ of 0.05 mg/L. The in-life phase was initiated after groups of 60 fertilised eggs were distributed evenly between four replicate vessels per treatment. Each group of 15 eggs was placed into a fry chamber suspended in each test vessel. After 14, 21, 28 and 35 days post fertilisation (pf), the fish larvae were digitally photographed. Post hatch survival rates were estimated by evaluating photographs using electronically supported counting and analysis. At test end, fish lengths and group dry weights of replicate fish groups were measured.

At test start and weekly thereafter, samples of the test media were taken for chemical analysis. The chemical analyses of the test media revealed mean concentrations for the single test vessels between 81 and 123 % of the nominal concentrations. The effect data evaluation was based on the mean measured concentrations. The mean concentrations per treatment were found to be between 93.5 and 103 % of the nominal values and were calculated to be 0.123, 0.256, 0.509, 0.939 and 1.870 mg/L. In addition to the chemical analysis of the test item, the concentrations of carbendazim in the different treatments were measured. For carbendazim the mean concentrations per treatment were found to be between 6.3 and 21.0 % of the nominal values of carbendazim and were calculated to be 0.0146, 0.0209, 0.0281, 0.0442 and 0.0699 mg/L.

Hatch of the fish larvae was not affected by the test item. The hatching success was > 95 % at all test concentrations of the test item. The evaluation of post hatch survival revealed a significant decrease at 0.939 mg/L and 1.870 mg/L after 28 and 35 days (test end). Statistically significant reduced post hatch survival at the low treatment levels 0.256 mg/L and 0.509 mg/L was only observed after 14 and 21 days pf. The respective rates of post hatch survival thereafter were almost comparable to those of after 21 days pf, but no statistically significant differences to the control were found at all later observation dates. Based on this observation, the decreased survival at 0.256 and 0.509 mg/L would not be considered as biologically relevant.

An EC₁₀ value was calculated for this endpoint. The probit analysis revealed an EC₁₀ of 0.390 mg/L (confidence limits: 0.147 - 0.645 mg/L). Since the fit of the regression curve was based on an effect range limited to a maximum of 33 % compared to control observed at the highest treatment level, the confidence limits were accordingly wide and spanned a range over two test concentrations.

Fish lengths were recorded on day 35 (test end). The single dry weights were calculated by dividing the group dry weights by the number of remaining fish on day 35 pf. Fish lengths and the single dry weights were found to be not significantly inhibited by the test item. It should be noticed, that an increase of both lengths and weights was observed at all treatments of the test item, which was not concentration dependent. The single dry weight was between 1.7 to 2.0 times higher than the single dry weight of the control. The observed increase in growth was not considered as negative effect on the fish. In addition, no abnormality in the fish behaviour was observed.

No adverse effect could be observed on the fish larvae concerning the endpoint hatching success. Post hatch survival was decreased by the test item at mean measured concentrations between 0.939 mg/L and 1.870 mg/L at test end. Lengths and single dry weights were found to be elevated at all investigated test concentrations. However, this was not considered as negative effect on the test organisms.

Thus, the lowest no effect concentration, based on the mean measured concentrations, was determined to be 0.509 mg/L for the endpoint post hatch survival after 35 days. The EC₁₀ for post hatch survival was calculated to be 0.39 mg/L (CL: 0.147 – 0.645 mg/L).

Description of key information

Fish Early Life Stage (FELS) toxicity test (OECD 210)

NOEC = 0.509 mg/L

EC₁₀ = 0.390 mg/L (CL: 0.147 – 0.645 mg/L)

(35-d, EC₁₀ > NOEC based on confidence limit CL: 0.147 – 0.645 mg/L)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.39 mg/L

Additional information

For chronic toxicity to fish, two long-term studies are available: OECD 210 (FELS) and OECD 204 (long-term toxicity to fish). Since OECD 210 covers the life cycle of the fish, this study is the most preferred study by ECHA and will be used for risk assessment in this dossier.

 

Fish Early Life Stage (FELS) toxicity test

The toxicity of the test item to fish early life stages was determined according to OECD 210. For this purpose, freshly fertilised eggs of zebrafish (Danio rerio) (less than 4 hours of age, post fertilisation) were exposed to 0.125, 0.25, 0.5, 1 and 2 mg/L. the lowest no effect concentration, based on the mean measured concentrations, was determined to be 0.509 mg/L for the endpoint post hatch survival after 35 days. The EC₁₀ for post hatch survival was calculated to be 0.390 mg/L (CL: 0.147 – 0.645 mg/L).

 

Long-term toxicity to fish

In a prolonged toxicity (28-days) of the test item to Rainbow trout (Salmo gairdneri) according to OECD 204 in a flow through test system. Fresh stock solutions of the test item dissolved in 1 % Tween 80-dimethylformamide were prepared daily for dispensing by the continuous flow apparatus. The test concentrations were 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The LC₅₀ was determined to be 0.60 mg/L and the NOEC was determined to be 0.32 mg/L.