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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 8th June 2015 Experimental completion date: 2nd July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations
Water samples were taken from the control and all surviving test groups at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.45, 1.0, 2.2, 4.5 and 10 mg/L.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 10 June 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 15 to 22 June 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 C with a dissolved oxygen content of greater than or equal to 9.3 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There were two mortalities in the 7 days prior to the start of the test (0.6% of population) and the fish had a mean standard length of 4.0 cm (sd = 0.2) and a mean weight of 0.77 g (sd = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.27 g bodyweight/liter (static volume).

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
total hardness of approximately 140 mg/L as CaCO3
Test temperature:
approximately 14 C
pH:
7.7 - 8.3
Dissolved oxygen:
9.4 - 10.2 mg O2/L
Salinity:
Not applicable - freshwater study
Nominal and measured concentrations:
In the range-finding test fish were exposed to a series of nominal test concentrations of 0.10, 1.0, and 10 mg/L

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.45, 1.0, 2.2, 4.5 and 10 mg/L.
Details on test conditions:
As in the range-finding test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 13 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Initial Test
After approximately 21 hours of exposure all seven fish exposed to a concentration of 9.5 mg/L were observed to be dead, therefore, the test was terminated. These results indicate that the LC50 was less than the threshold concentration, therefore, a full acute toxicity test including a range-finding test was conducted.

Range-finding Test
The results showed no mortalities at the test concentrations of 0.10 and 1.0 mg/L, however, 100% mortality was observed at 10 mg/L.

After approximately 6 hours exposure 1 out of 3 fish at 10 mg/L was observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality at this time point.

Based on this information test concentrations of 0.45, 1.0, 2.2, 4.5 and 10 mg/L were selected for the definitive test.

Definitive test
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 1.9 mg/L, the lowest test concentration resulting in 100% mortality to be 8.6 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 3.8 mg/L. The No Observed Effect Concentration (NOEC) was 1.9 mg/L.

Initial Test

After approximately 21 hours of exposure all seven fish exposed to a concentration of 9.5 mg/L were observed to be dead, therefore, the test was terminated. These results indicate that the LC50 was less than the threshold concentration, therefore, a full acute toxicity test including a range-finding test was conducted.

Range-finding Test

The results showed no mortalities at the test concentrations of 0.10 and 1.0 mg/L, however, 100% mortality was observed at 10 mg/L.

 

After approximately 6 hours exposure 1 out of 3 fish at 10 mg/L was observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality at this time point.

 

Based on this information test concentrations of 0.45, 1.0, 2.2, 4.5 and 10 mg/L were selected for the definitive test.

 

Chemical analysis of the freshly prepared test preparations at 0 hours showed measured test concentrations to range from 82% to 95% of nominal concentrations. Analysis of the old test preparations at 24 hours showed measured test concentrations to range from 69% to 84% of nominal concentrations indicating that the test item was unstable under test conditions.

Definitive Test

Verification of Test Concentrations

The geometric mean measured test concentrations were determined to be: 

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

Expressed as a % of the Nominal Test Concentration

0.45

0.32

71

1.0

0.74

74

2.2

1.9

86

4.5

3.8

84

10

8.6

86

 

Mortality Data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 3 and the relationship between percentage mortality and concentration at 96 hours is given in Figure 1.

 

Analysis of the mortality data by the Probit analysis using Linear Maximum-Likelihood regression at 24, 48, 72 and 96 hours based on the geometric mean measured test concentrations gave the following results: 

Time (h)

LC50 (mg/L)*

3

>10

6

>10

24

4.3

48

3.9

72

3.7

96

3.7

 

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 1.9 mg/L, the lowest test concentration resulting in 100% mortality to be 8.6 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 3.8 mg/L. The No Observed Effect Concentration (NOEC) was 1.9 mg/L. 

 

Sub-Lethal Effects

Sub-lethal effects of exposure were observed at test concentrations of 3.8 mg/L and above. These responses were loss of equilibrium, sitting/swimming at the bottom, increased pigmentation and moribund (see Table 4).

 

After approximately 47 hours exposure 1 out of 5 fish at 3.8 mg/L was observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) this fish was killed and classed as a mortality for the following observational time point.

 

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was equal to or greater than 60% of ASV (6.1 mg O2/L) in the control and test vessels.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

*95% confidence limits could not be calculated as data did not fit available models

Validity criteria fulfilled:
yes
Conclusions:
Exposure ofrainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the geometric mean measured test concentrations: 

Time Point LC50 No Observed Effect Concentration (NOEC) Lowest Observed Effect Concentration (LOEC)
(Hours) (mg/L) (mg/L) (mg/L)
96 3.7* 1.9 3.8
*95% confidence limits could not be calculated as data did not fit available models
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 

Methods

In accordance with the recommendations of REACh, a test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity toDaphnia magnastudy is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The ErC50 value obtained from theAlgal Growth Inhibition study (Study Number41500373) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 9.5 mg/L.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 9.5 mg/L at a temperature of 14 to 15 °C under static test conditions. After approximately 21 hours of exposure all seven fish exposed to a concentration of 9.5 mg/L were observed to be dead, therefore, the test was terminated. These results indicate that the LC50was less than the threshold concentration, therefore, a full acute toxicity test including a range-finding test was conducted.

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test item over a range of concentrations of 0.45, 1.0, 2.2, 4.5, and 10 mg/L for a period of 96 hours at a temperature of 14 to 15 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

Results

Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations to range from 78% to 96% of nominal concentrations. Analysis of the old test preparations at 24 and 96 hours showed measured test concentrations to range from 60% to 83% of nominal concentrations. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations to give a "worst case" analysis of the data.

Exposure ofrainbow trout (Oncorhynchus mykiss)to the test item gave the following results based on the geometric mean measured test concentrations: 

Time Point

(Hours)

LC50
(mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

96

3.7*

1.9

3.8

*95% confidence limits could not be calculated as data did not fit available models

Description of key information

For fish, the 96 -h LC50 value is 3.7 mg/L as determined from a study according to OECD TG 203 and in compliance with GLP criteria.

Key value for chemical safety assessment

LC50 for freshwater fish:
3.7 mg/L

Additional information

The short-term toxicity to freshwater fish was determined in a study according to OECD TG 203 and in compliance with GLP criteria. In this study, groups of 7 rainbow trout (O. mykiss) were exposed to nominal concentrations of 0 (control), 0.45, 1.0, 2.2, 4.5 and 10 mg/L for a period of 96 hours under semi-static test conditions (daily renewal). Nominal test concentrations were verified by a validated analytical method. The measured concentrations in the fresh media were between 78 and 97% of nominal but declined to 74-85% of initial concentrations in the 24 hours old media. Effect values were therefore expressed as geomean measured concentrations (0.32, 0.74, 1.9, 3.8 and 8.6 mg/L for nominal concentrations of 0.45, 1.0, 2.2, 4.5 and 10 mg/L, respectively). Incidences of mortality and sub-lethal effects were recorded after 3, 6, 24, 48, 72 and 96 hours exposure. All OECD validity criteria were met.

Mortality was first observed at the test concentration of 3.8 mg/L and cumulated to 57% after 96 hours exposure. At the highest test concentration of 8.6 mg/L cumulative mortality reached 100% within 24 hours after the start of the test. Sub-lethal effects of exposure were observed only at the two highest concentrations tested and consisted of increased pigmentation, sitting/swimming at the bottom and moribund at 3.8 mg/L and increased pigmentation, loss of equilibrium and sitting at the bottom at 8.6 mg/L. Based on these findings the 96-h LC50 value is determined at 3.7 mg/L.