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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 April 2018 - 23 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
November 2018
Specific details on test material used for the study:
Appearance: Light yellow powder
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Range-finding test: samples of fresh test preparations were taken on days 0 and 2 and of the expired test preparations on days 2 and 5 and stored frozen prior to analysis.

Definitive test: The concentration and stability of the test item in the test preparations (control and all surviving test groups) were verified by chemical analysis on days 0, 6, 13, 20, 27 and 32 (fresh media) and for old media on days 1, 7, 14, 21, 28 and 33 (replicates 1 to 4 pooled).
Vehicle:
no
Details on test solutions:
An auxiliary solvent (Dimethylformamide, DMF) was used during a preliminary range-finding test, however prior to the definitive test it was determined that similar concentration levels could be achieved without the use of the auxiliary solvent, therefore the definitive test was performed without this solvent.
A nominal amount of test item (7.5mg) was dissolved in test water with the aid of magnetic stirring for 20 minutes, and the volume adjusted to 20 liters to give a 0.36 mg a.i./L stock solution. A series of dilutions were made from this stock solution to give test solutions of 0.0225, 0.045, 0.090 and 0.18 mg a.i./L.
On days 0 to 13 each test solution was inverted several times to ensure adequate mixing and homogeneity. On days 14 to 32 each test solution was stirred using a magnetic stirrer to ensure adequate mixing and homogeneity.
Test organisms (species):
Pimephales promelas
Details on test organisms:
- Common name: fathead minnows (Pimephales promelas), in-house culture
- Age at study initiation: newly laid fathead minnow eggs
- Feeding during test : Brine shrimp nauplii from day 6 to day 10 and freshly hatched basic grade brine shrimp nauplii from day 11 to day 32
- Breeding: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
Included 28 days post-hatching
Post exposure observation period:
At the end of the test only the length and wet weight of each surviving fish was determined
Hardness:
124 - 156 mg/L as CaCO3 at start of the test; 132 - 160 mg/L at termination
Test temperature:
23.6 - 25.3 °C
pH:
6.8 - 9.0
Dissolved oxygen:
> 5.1 mg O2/L (62% Air saturation value)
Nominal and measured concentrations:
Nominal concentrations: 0, 0.0225, 0.045, 0.090, 0.18, 0.36 mg/L
Average geometric mean measured concentrations: 0, 0.0051, 0.0088, 0.021, 0.036, 0.043 mg/L. Since the concentrations were not stable between renewals, the effect parameters were based on the geometric mean measured concentrations.
Details on test conditions:
The range-finding test was conducted in 1 liter glass vessels each containing approximately 400 mL of test preparation from day 0 to 6, increasing to approximately 800 mL from day 7 to the end of the test. Two replicate flasks were used for each control and test concentration. Twenty eggs were placed into each replicate test vessel at the start of the test. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 15 days under semi-static test conditions. The control group was maintained under identical conditions but not exposed to the test item.

Based on the results of a preliminary range-finding test, newly laid fathead minnow eggs (four replicates of 20 eggs per group) were exposed to solutions of the test item at nominal concentrations of 0.0225, 0.045, 0.090, 0.18 and 0.36 mg a.i./L for a period of 33 days under semi-static 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 glass 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 14 to day 33. Four replicate flasks were used for each control and test concentration.
A semi-static test regime was employed in the test involving renewal of the test preparations daily throughout the test, with the exception of day 3 where there was no water change to avoid causing premature hatching of the eggs.
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 maximum deviation of ±1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness and 20 minute dawn and dusk transition periods for a period of 33 days.
The test vessels were aerated via narrow bore glass tubes from day 8 onwards. The eggs and larvae were not individually identified.
The larvae were fed freshly hatched special grade brine shrimp nauplii from day 6 to day 10 and freshly hatched basic grade brine shrimp nauplii from day 11 to day 32.

- Test water: laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Post-hatch survival
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Post-hatch survival
Key result
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Post-hatch survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.021 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
0.036 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
EC50
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
0.021 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
EC50
Effect conc.:
> 0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Some larvae were observed to be small or pale. These observations were recorded from day 8 or day 12 respectively in 0.0088 mg/L replicate R3 (one larva), 0.0088 mg/L replicate R4 (two larva). The overall number of these observations was very low (five in total) and were not concentration dependent. In addition, one larva in control replicate 2 was observed with a bent spine from day 16 to 25. As there were no sub lethal effects noted at the 0.0051, 0.021, 0.036 and 0.043 mg/L test concentrations, the observations noted are considered to be due to normal biological variation, and not attributable to the test material.

The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed. Mean hatching rate was 96-99%. Mean survival rate was 92-100%.
Results with reference substance (positive control):
none
Reported statistics and error estimates:
The LCx value for the number of eggs hatching were calculated by Probit analysis using Linear Maximum-Likelihood regression and Chi2 2x2 Table Test with Bonferroni Correction..
The LCx values for post-hatch survival were calculated by the Weibull analysis using Linear Maximum-Likelihood regression and Chi2 2x2 Table Test with Bonferroni Correction.
The ECx values and associated confidence limits for length and wet weight at the end of the test were calculated by the 3-parameter normal Cumulative Distribution Function and Williams Multiple Sequential t-test.
All EC/LCx determinations were performed using the ToxRatPro software package (TOXRAT, 2015).

Table 1

Endpoint

Concentration mg a.i./L

(95% confidence limits)

Number Hatched

LC10

>0.043 (Not determined)

LC20

>0.043 (Not determined)

LC50

>0.043 (Not determined)

NOEC

0.043

LOEC

>0.043

Post-hatch Survival

LC10

>0.043 (Not determined)

LC20

>0.043 (Not determined)

LC50

>0.043 (Not determined)

NOEC

0.043

LOEC

>0.043

Body Length

EC10

>0.043 (Not determined)

 

EC20

>0.043 (Not determined)

 

EC50

>0.043 (Not determined)

 

NOEC

0.021

 

LOEC

0.036

Body Weight

EC10

0.031 (0.007 – 0.136)

 

EC20

>0.043 (Not determined)

 

EC50

>0.043 (Not determined)

 

NOEC

0.0088

 

LOEC

0.021

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
Exposure of fathead minnow larvae to the test item gave the following values:
- Number hatched and post-hatch survival: NOEC 0.043, LOEC >0.043, LC50 >0.043 mg/L
- Body length: NOEC 0.021, LOEC 0.036, EC50 >0.043 mg/L
- Body weight: NOEC 0.0088, LOEC 0.021, EC50 >0.043 mg/L.
Executive summary:

A semi-static fish early-life stage toxicity test was performed in order to assess possible lethal and sub-lethal effects during the embryonic and early larval development of the fathead minnow (Pimephales promelas).

The study was conducted in accordance with OECD 210 and in compliance with GLP. Fertilized eggs were exposed for 33 days to an untreated control and the test item at average geometric mean measured concentrations of 0.0051, 0.0088, 0.021, 0.036 and 0.043 mg/L (80 eggs per group, divided into four replicates).

Embryonic and larval survival was not affected in any of the tested concentrations (NOEC 0.043, LOEC >0.043, LC50 >0.043). Body weight was significantly affected at 0.036 mg/L (LOEC; NOEC 0.021 mg/L). Body weight was significantly affected at 0.021 mg/L (LOEC; NOEC 0.0088 mg/L). EC50 values for both body weight and body length was >0.043 mg/L.

The study met all validity criteria and is considered valid without restriction.

Description of key information

Fish early life stage (Fathead minnow, Pimephales Promelas), most sensitive endpoint: body weight

NOEC = 0.0088 mg/L

LOEC = 0.021 mg/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.009 mg/L

Additional information

A semi-static fish early-life stage toxicity test was performed in order to assess possible lethal and sub-lethal effects during the embryonic and early larval development of the fathead minnow (Pimephales promelas).

The study was conducted in accordance with OECD 210 and in compliance with GLP. Fertilized eggs were exposed for 33 days to an untreated control and the test item at average geometric mean measured concentrations of 0.0051, 0.0088, 0.021, 0.036 and 0.043 mg/L (80 eggs per group, divided into four replicates).

Embryonic and larval survival was not affected in any of the tested concentrations (NOEC 0.043, LOEC >0.043, LC50 >0.043). Body weight was significantly affected at 0.036 mg/L (LOEC; NOEC 0.021 mg/L). Body weight was significantly affected at 0.021 mg/L (LOEC; NOEC 0.0088 mg/L). EC50 values for both body weight and body length was >0.043 mg/L.

The study met all validity criteria and is considered valid without restriction.