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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October 2017 - ****
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Physical state/Appearance: Light yellow to tan solid
Purity: UVCB substance
Analytical monitoring:
yes
Details on sampling:
Samples were take from the control and 100 mg/L loading rate WAF test group from the freshly prepared bulk test preparation on Days 0, 4, 8, 13, 18 and 25 and from the old or expired media on Days 1, 6, 11, 15, 20, 27 and 32 for quantitative analysis. The samples were either stored frozen prior to analysis. The test samples were thawed with the aid of a water bath then diluted with tetrahydrofuran.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test material, the test solutions were prepared as a Water Accommodated Fraction (WAF). A nominal amount of test material was added to the surface of test water to give the 100 mg/L loading rate. After the addition of the test material, the test water was stirred by magnetic stirrer at a rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and removal of the WAF by filtering through a glass wool plug (2 to 4 cm in length) was justified. Microscopic observations of the WAF were performed after filtering and showed no particles or micro-dispersions of test material to be present. The control group was prepared similarly, but was not exposed to the test material. After dosing the control and 100 mg/L loading rate were observed to be clear, colorless solutions by visual inspection.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: Envigo Research Limited
- Age at study initiation (mean and range, SD): Freshly fertilized eggs, confirmed to be at the early blastodisc stage
- Method of breeding: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized
- Feeding: Breeding stock fish were fed ZM 400 flake food daily. Larvae were fed <24 hour old special grade brine shrimp nauplii from Day 6 to Day 10 (post hatch), and 24 hour old basic grade brine shrimp nauplii from Day 11 (post hatch) to the end of the test.

Test type:
semi-static
Water media type:
freshwater
Remarks:
Laboratory tap water that was dechlorinated by passage through an activated carbon filter, partly softened, then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Limit test:
yes
Total exposure duration:
32 d
Hardness:
142 to 156 mg/L - Start of test
128 to 140 mg/L - End of test
Test temperature:
25 to 26 °C
pH:
6.8 to 8.6
Dissolved oxygen:
5.1 mg/L
Nominal and measured concentrations:
Based on the results of the range-finding test, which was consucted using nominal loading rates of 1.0, 10 and 100 mg/L, the definitive test was conducted at a single nominal loading rate of 100 mg/L.

Details on test conditions:
In the definitive test, 1 liter glass vessels were used from day 0 to Day 13 and, from Day 14 to the end of the test, 5 liter gass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 5, 800 mL from Day 6 to Day 13 and 4000 Ml from Day 15 to Day 32. Four replicae flasks were used for each control and test concentration.

A semi-static test regime was employed in the test involving a renewal of the test preparations three times per week from the start of the test.

Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25°C with a maximim deviation of +/-1.5°C between test chambers or between successive days, and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods throughout the 32-day exposure period.

The test vessels were aerated via narrow bore glass tubes from Day 13 onwards. The eggs and larvae were not individually identified.

OTHER TEST CONDITIONS
Dissolved oxygen concentrations and pH were recorded before and after each test media renewal. Oxygen levels fell outside the specified range of 60 - 100% ASV and the grade and age of brine shrimp fed to the juvenile fish deviated slightly from the guideline. These deviations were considered not to have adversely impacted the study. The water temperature and light intensity were recorded daily throughout the test. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24 hour old test preparations. Surplus food and faeces were removed regularly from the test vessels.

Reference substance (positive control):
no
Key result
Duration:
32 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatchability, post-hatch survival, growth (length and weight)
Details on results:
RANGE-FINDING TEST

There were no differences in hatching, survival and growth between the control and any of the nominal loading rates tested. A single fish in the 10 mg/L loading rate test group was observed to be pale on Days 7 and 8 and one was observed to be small on Days 11 to 14. Given that this was not a concentration dependent response, this response was not considered to be treatment related and as such had no impact on the outcome of the test.

DEFINITIVE TEST

The start of egg hatching and the completion of hatching were observed on Day 4 of the test. Mean hatching rate in the control and 100 mg/L loading rate groups was 98%. Mean post-hatch survival rate in the control and 100 mg/L loading rate groups was 91% and 99%, respectively.

The group mean length and weight in the control group were 20.92 mm and 73.5 mg, respectively. The group mean length and weight in the 100 mg/L loading rate group were 20.72 mm and 75.8 mg, respectively.

A single fish in the control group was observed to be small on Days 16 to 22 and Days 25 to 29, and two control fish were observed to be small on Days 23 and 24. These responses were considered to be due to natural variation based upon historical control data and as such were considered not to have had an impact on the outcome of the test.
Reported statistics and error estimates:
Analysis of the number of eggs successfully hatching and the post-hatch survival were compared using the Fishers Exact Binomial Test. No significant differences (P>/=0.05) were found.

Analysis of the fish length and weight data obtained at termination of the test were compared using Two-sample t-test procedure. No significant differences (P>/=0.05) were found.

Cumulative Number of Hatched (Live) Larvae in the Definitive Test

Nominal Loading Rate
(mg/L)

Number of Live Larvae (Cumulative)

Day

1

2

3

4

5

6

7

8

9

10

11

Control

R1

0

0

0

20

20

19

19

19

19

19

19

R2

0

0

0

20

20

20

20

20

20

20

20

R3

0

0

0

18

18

18

18

18

18

18

18

R4

0

0

0

19

19

19

19

19

19

19

19

100

R1

0

0

0

20

20

20

20

20

20

20

20

R2

0

0

0

19

19

19

19

19

19

19

19

R3

0

0

0

20

20

20

20

20

20

20

20

R4

0

0

0

18

18

18

18

18

18

18

18

Nominal Loading Rate
(mg/L)

Number of Live Larvae (Cumulative)

Day

12

13

14

15

16

17

18

19

20

21

22

Control

R1

19

19

19

19

18

18

18

18

18

18

18

R2

20

20

20

20

20

20

20

20

20

20

20

R3

18

18

18

18

18

18

18

18

18

18

18

R4

19

18

18

18

18

18

18

18

18

18

18

100

R1

20

20

20

20

20

20

20

20

20

20

20

R2

19

19

19

19

19

19

19

19

19

19

19

R3

20

20

20

20

20

20

20

20

20

20

20

R4

18

18

18

18

18

18

18

18

18

18

18

Nominal Loading Rate
(mg/L)

Number of Live Larvae (Cumulative)

Day

23

24

25

26

27

28

29

30

31

32

Control

R1

18

18

18

16

16

16

16

16

16

16

R2

20

20

20

20

20

20

20

20

20

20

R3

18

18

17

17

17

17

17

17

17

17

R4

18

18

18

18

18

18

18

18

18

18

100

R1

20

20

20

20

20

20

20

20

20

20

R2

19

19

19

19

19

19

19

19

19

19

R3

20

20

20

20

20

20

20

20

20

20

R4

18

18

18

18

18

18

18

18

18

18


R          =    Replicate

Validity criteria fulfilled:
yes
Conclusions:
The exposure of newly fertilized eggs of fathead minnows to the test material was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

A study was conducted in accordance with OECD Chemicals Testing Guideline No. 210 to assess the effects of the test material to the freshly hatched larvae of the fathead minnow. Due to the low aqueous solubility and complex nature of the test material, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a single WAF of the test material, prepared at 100 mg/L loading rate, for a period of 32 days at a temperature of approximately 25°C under semi-static test conditions. The number of mortalities and any sub lethal effects of exposure in each test and control vessel were recorded until termination of the test (28 days post-hatch).

Analysis of the fresh test preparations on Days 0, 4, 8, 13, 18 and 25 showed measured test concentrations ranged from less than the limit of quantification (LOQ, 0.0078 mg/L) of the analytical method to 0.038 mg/L. Analysis of the old or expired test preparations on Days 1, 6, 11, 15, 20, 27 and 32 showed measured concentrations from less than the LOQ to 0.028 mg/L.

The exposure of newly fertilized eggs of fathead minnows to the test material was determined to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. 

Description of key information

In an OECD 210 study, conducted according to GLP, exposure of newly fertilized eggs of the fathead minnow, Pimpephales promelas, to the test material under semi-static conditions was determined to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate (NOELR) was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information