5-bromo-1,3-dichloro-2-fluorobenzene

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: 05 July 2022, experiment end: 03 August 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

SEPA, P.R. China, the Guidelines of Chemical Testing Good Laboratory Practices (HJ/T 155-2004), China is not a member of OECD and hence there is no agreement on mutual acceptance of data

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

SEPA, P.R. China, the Guidelines of Chemical Testing Good Laboratory Practices (HJ/T 155-2004), China is not a member of OECD and hence there is no agreement on mutual acceptance of data

Analytical monitoring:

yes

Details on sampling:

Samples of test solutions for analysis were taken on Day 0, 9 and 16 for new test solutions and on Day 6, 13 and 20 for old solutions.

Vehicle:

no

Details on test solutions:

A stock solution with a nominal concentration of 18.2 mg/L was prepared by transferring 10 µL into a 1 L volumetric flask and adding test water to the mark. This was subsequently diluted with test water to obtain the different test concentrations.

Test organisms (species):

other: Rare minnow (Gobiocypris rarus)

Details on test organisms:

Fish were reared in the test facility and eggs used in the study were collected on 06 July 2022. The stage of fish eggs at the beginning of the test was the two-cell stage, which was confirmed by microscopy.
Post-hatching feeding: Food for the fish was frozen brine shrimps. First feeding time was seven days after test initiation. The diet was provided two times per day; one drop of brine shrimps was fed each time. Surplus food and feces were removed after a period of 1.5 hours following feeding.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

22 d

Hardness:

Start: 238 to 242 mg/L (as CaCO3)
End: 185 to 190 mg/L (as CaCO3)

Test temperature:

24.2 to 25.8 °C

pH:

7.32 to 8.06

Dissolved oxygen:

6.07 to 8.52 mg/L

Nominal and measured concentrations:

Nominal: 0.1, 0.316, 1, 3.16 and 10 mg/L
Measured geometric mean: 0.044, 0.12, 0.427, 1.4, 4.11 mg/L

Details on test conditions:

Test vessel: 1-L wide-mouth bottles with cover
Test water volume: 1 L in each vessel
Medium replacement: test medium was replaced only once during the first week and twice the week thereafter, about 3/4 of the test solutions were exchanged every time
Loading: 20 eggs per vessel
Light cycle: 12 hours light, 12 hours darkness
A blank control was included (four replicates). Similarly, four replicates were used in each exposure group.

Reference substance (positive control):

no

Key result

Duration:

22 d

Dose descriptor:

NOEC

Effect conc.:

0.044 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

Hatchabillity and survival in controls: the hatchability was 83.7% and the survival rate was 100%, which met the requirements of >66% for hatchability of fertilised eggs and >70% for survival rate.
The test substance had a dose-dependent effect on the hatchability of fertilised eggs and the survival rate. The hatching success was 81.2% (0.1 mg/L), 81.2% (0.316 mg/L), 61.3% (1 mg/L), 57.5% (3.16 mg/L) and 0% (10 mg/L). The survival rate was 100% (0.1 mg/L), 95.4% (0.316 mg/L), 95.4% (1 mg/L), 95.9% (3.16 mg/L) and 0% (10 mg/L).
Exposure to the substance had a dose-dependent adverse effect on the weight and length of surviving fish at the end of the study.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The NOEC was calculated with the Step-down Cochran-Armitage test (for hatchability on Day 6), Bonferri Chi test (for survival on Day 22), Williams Multiple Sequential t-test (for fresh weight and length).

Table 1: Hatching success

Conc. (mg/L)	Replicate	Day 1		Day 2		Day 3		Day 4		Day 5		Day 6		Hatchability (%)
		Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead
Blank control	1	0	4	0	4	7	4	16	4	16	4	16	4	83.7
	2	0	2	0	2	11	2	18	2	18	2	18	2
	3	0	2	0	2	10	2	16	3	16	3	17	3
	4	0	4	0	4	11	4	16	4	16	4	16	4
0.1	1	0	2	0	2	6	3	15	3	16	4	16	4	81.2
	2	0	4	0	4	12	4	15	4	16	4	16	4
	3	0	4	0	4	10	4	16	4	16	4	16	4
	4	0	3	0	3	14	3	17	3	17	3	17	3
0.316	1	0	3	0	3	10	4	16	4	16	4	16	4	81.2
	2	0	4	0	4	12	4	15	4	16	4	16	4
	3	0	4	0	4	8	4	15	4	16	4	16	4
	4	0	3	0	3	9	3	16	3	17	3	17	3
1	1	0	5	0	7	10	7	11	8	12	8	12	8	61.3
	2	0	3	0	7	11	7	11	8	12	8	12	8
	3	0	1	0	5	8	9	9	10	10	10	10	10
	4	0	1	0	3	10	4	12	5	15	5	15	5
3.16	1	0	6	0	8	6	10	7	10	8	10	10	10	57.5
	2	0	6	0	9	5	10	10	11	9	11	9	11
	3	0	1	0	3	9	4	13	5	15	5	14	6
	4	0	2	0	3	7	5	15	5	15	5	13	7
10	1	0	3	0	3	0	15	0	20	/	/	/	/	0
	2	0	1	0	4	0	10	0	20	/	/	/	/
	3	0	1	0	3	0	11	0	20	/	/	/	/
	4	0	1	0	3	0	13	0	20	/	/	/	/

Table 2: Body weight and length of test fish at the end of the study

Conc. (mg/L)	Replicate	Dish weight (g)	Dish weight with fish (g)	Number of fish	Mean weight per fish (mg)	Average length of fish (mm)
Blank control	1	41.5823	41.6112	16	1.8063	6.2012
	2	45.2717	45.2991	18	1.5222	5.9482
	3	45.8078	45.8356	17	1.6353	6.2380
	4	26.4102	26.4357	16	1.5938	6.0778
0.1	1	48.2140	48.2366	14	1.6143	6.3654
	2	47.8095	47.8324	15	1.5267	6.2143
	3	38.8244	38.8466	16	1.3875	6.1499
	4	47.4723	47.4950	17	1.3353	6.1781
0.316	1	42.5490	42.5654	16	1.0250	5.9109
	2	45.9190	45.9416	16	1.4125	5.9359
	3	44.3430	44.3577	15	1.0933	5.7921
	4	42.5925	42.6132	15	1.3800	5.9054
1	1	50.8393	50.8533	12	1.1667	5.9007
	2	36.2375	36.2532	12	1.3083	5.9670
	3	47.6531	47.6609	9	0.8667	5.4750
	4	47.3552	47.3552	14	1.1000	5.8463

Validity criteria fulfilled:

yes

Conclusions:

The test substance demonstrated long-term toxicity to rare minnow over a period of 22 days in this fish early-life stage study. The lowest NOEC in the study was found to be 0.044 mg/L based on the wet weight of fish.

Executive summary:

A fish early-life stage toxicity study was conducted with the substance according to OECD TG 210 (version 2013). The study was conducted under semi-static conditions with rare minnow (Gobiocyprus rarus) over a period of 22 days. Following a range-finding study, the definitive study employed five test concentrations: 0.1, 0.316, 1, 3.16 and 10 mg/L. A blank control was performed concurrently under the same test conditions. Results of the chemical analysis showed that the measured concentrations were less than 80% of the nominal concentrations. Thus, the geometric means of the measured concentrations were used to calculate the effect levels in the study. Blank control and exposure groups consisted of four replicates and 20 fertilised eggs (initially at the two-cell stage) were introduced into each replicate. Studies were conducted with a test water volume of 1 L.

The test solutions were changed once during the first week, and twice a week during the remainder of the study. The pH value, dissolved oxygen and temperature of new and old solutions were determined once or twice a week. The hardness of the test medium was measured at the start and the end of the study. The hatching success of fertilised eggs in the controls was 83.7% exceeding the validity criterion of 66%. Furthermore, the suviving rate in the controls was 100, meeting the validity criterion of at least 70% survival.

Exposure to the test substance had an adverse, dose-dependent effect on the hatching success of fertilised eggs, the survival rate and the weight and length of surviving fish at the end of the study. The most sensitive endpoints were the weight and length of surviving fish at the end of the study. The corresponding 22-day NOEC was determined to be 0.044 mg/L. 

Description of key information

22-day NOEC = 0.044 mg/L in rare minnow (Gobiocypris rarus), semi-static (China GLP, OECD TG 210)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.044 mg/L

Additional information

Two aquatic toxicity studies were performed with the substance to test the potential long-term effects on the freshwater fish rare minnow (Gobiocypris rarus). These studies were conducted to meet the data requirements for registration in a non-EEA country. In a fish early life-stages study conducted over a period of 22 days and following in principle OECD TG 210, the lowest NOEC of 0.044 mg/L was observed for the wet weight of fish at the end of the exposure period. In a second study on the embryo and sac-fry stages over a period of 9 days following in principle OECD TG 212, the lowest NOEC of 0.284 mg/L was observed for abnormal appearance or behaviour of larvae. Both studies showed that the substance is expected to have long-term adverse effects on freshwater fish.