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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:
The study was conducted between 20 June 2011 and 15 July 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Reason / purpose:
reference to other 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:
- Concentrations:
In the definitive test a single concentration of 100 mg ai/l was used.

- Sampling method:
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. 

- Sample storage conditions before analysis:
The 0 and 24 hour samples were stored at approximately -20°C prior to analysis.
Vehicle:
no
Details on test solutions:
Range-finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test fish were exposed to a nominal test concentration of 100 mg active ingredient (ai)/l. A single test concentration was used as results from the range-finding test conducted for the Acute Toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 41101845) indicated that toxicity was not expected at this concentration. The test item was dissolved directly in dechlorinated tap water. At the request of the Sponsor the test concentrations were corrected for a water content of 4.3%.
An amount of test item (2086 mg) was dissolved in dechlorinated tap water and the volume adjusted to 500 ml to give a 4000 mg ai/l stock solution. The entire volume of this stock solution was diluted in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 100 mg ai/l test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 3 fish were added to each 20 litre test and control vessel and maintained at approximately 14°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 under static test conditions.
The control group was maintained under identical conditions but not exposed to the test item.
Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°prior to analysis.

Definitive test:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg ai/l to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation:
For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water.
An amount of test item (4180 mg) was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 4000 mg ai/l stock solution.
An aliquot (500 ml) of this stock solution was diluted in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 100 mg ai/l test concentration. This method of preparation was conducted in duplicate to give replicates R1 and R2.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Test Species
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 5 May 2011. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 29 June 2011 to 11 July 2011. 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 8.1 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 was less than 1% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.5 cm
(sd = 0.2) and a mean weight of 1.85 g (sd = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.65 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test Water
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) 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. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (see attached background material).


Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Hardness:
Total hardness of approximately 140 mg/l as CaCO3.
Test temperature:
The test vessels were sealed and maintained at approximately 14ºC.
pH:
The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter .
Please see Physico-Chemical Measurements appendix 4 (see any other information on results incl. tables section).
Dissolved oxygen:
The dissolved oxygen concentration was measured using a Hach HQ30d Flexi Handheld meter .
Please see Physico-Chemical Measurements appendix 4 (see any other information on results incl. tables section) for dissolved oxygen results.
Salinity:
Freshwater used.
Nominal and measured concentrations:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration (nominal) of 100 mg ai/I to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.
Details on test conditions:
Exposure conditions:
As in the range-finding test 20 litre glass exposure vessels were used for each 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 approximately 14ºC 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. Data from the control group was shared with similar concurrent studies.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Physico-chemical measurements:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits not stated
Details on results:
Range-finding Test:
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 (see any other information on results incl. tables section). There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the test concentration of 100 mg ai/l.
Chemical analysis of the 100 mg ai/l test preparation at 0 and 24 hours (see Appendix 3 - attached background material) showed measured concentrations of 99% and 102% of nominal value indicating that the test item was stable under test conditions.

Definitive Test:
Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 (see any other information on results incl. tables section).
There were no significant mortalities in 14 fish exposed to a test concentration of 100 mg ai/l for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LC50 (mg ai /l)
3 >100
6 >100
24 >100
48 >100
72 >100
96 >100

The results of the definitive test showed the No Observed Effect Concentration (NOEC) to be 100 mg ai/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

A single mortality was observed in the control and 100 mg ai/l replicate R1 after approximately 72 hours. This was considered to be due to natural causes and/or handling stress rather than a toxic effect given that no further mortality was observed.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg ai/l.

Sub-lethal effects:
There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentration of 100 mg ai/l for a period of 96 hours.

Observations on test item solubility:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements:
The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Verification of test concentrations
Analysis of the test preparations at 0, 24 and 96 hours (see Appendix 3 - attaced background material) showed measured test concentrations to range from 97% to 102% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Validation Criteria
The results of the test are considered valid given that:
-Only a single mortality was observed in the control group.
-The dissolved oxygen concentration at the end of the test was 96% to 98% of Air Saturation Values (ASV) in the control and test vessels.

Table 1              Cumulative Mortality Data in the Range-finding Test

Nominal

Concentration

(mg ai[1]/l)

Cumulative Mortality

(Initial Population = 3)

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Control

0

0

0

0

0

0

100

0

0

0

0

0

0

[*]ai = Active ingredient

Table 2              Cumulative Mortality Data in the Definitive Test

Nominal

Concentration

(mg ai*/l)

Cumulative Mortality
(Initial Population = 7)

%

Mortality

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96 Hours

Control

0

0

0

0

1**

1**

14**

100 R1

0

0

0

0

1**

1**

14**

100 R2

0

0

0

0

0

0

0


R1and R2= Replicates 1 and 2

* ai = Active ingredient

** Single mortality considered to be due to natural causes and/or handling stress rather than a toxic effect given that no further mortality was observed

Appendix 4      Physico-Chemical Measurements

Nominal

Concentration

(mg ai[*]/l)

Time (Hours)

0 Hours (Fresh Media)

24 Hours (Old Media)

24 Hours (Fresh Media)

pH

mg O2/l

%ASV**

TºC

pH

mg O2/l

%ASV**

T°C

pH

mg O2/l

%ASV**

T°C

Control

7.1

9.5

92

14

7.9

9.9

96

14

7.2

9.4

91

14

100 R1

7.4

9.6

93

14

7.7

9.9

96

14

7.4

9.7

94

14

100 R2

7.4

9.6

93

14

7.7

9.8

95

14

7.4

9.7

94

14

 

Nominal

Concentration

(mg ai*/l)

Time (Hours)

48 Hours (Old Media)

48 Hours (Fresh Media)

72 Hours (Old Media)

pH

mg O2/l

%ASV**

TºC

pH

mg O2/l

%ASV**

T°C

pH

mg O2/l

%ASV**

T°C

Control

8.0

9.9

96

14

7.1

9.8

95

14

8.1

10.3

100

14

100 R1

8.0

10.0

97

14

8.0

9.8

95

14

8.0

10.0

97

14

100 R2

7.9

9.9

96

14

8.0

9.8

95

14

8.0

10.1

98

14

 

Nominal

Concentration

(mg ai*/l)

Time (Hours)

72 Hours (Fresh Media)

96 Hours (Old Media)

pH

mg O2/l

%ASV**

TºC

pH

mg O2/l

%ASV**

T°C

Control

7.9

9.5

92

14

7.8

10.1

98

14

100 R1

8.0

9.5

92

14

8.1

9.9

96

14

100 R2

7.9

9.4

91

14

8.0

9.9

96

14


[*]ai = Active ingredient

**ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1and R2= Replicates 1 and 2

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg ai/l. The No Observed Effect Concentration was 100 mg ai/l.
Executive summary:

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.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of approximately 14ºC 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. At the request of the Sponsor all concentrations were corrected for a test item water content of 4.3%.

The 96-Hour LC50 based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 97% to 102% of nominal value and so the results are based on nominal test concentrations only.

Description of key information

In a single key study conducted in accordance with OECD 203, no effects were seen in the freshwater species Oncorhyncus mykiss (rainbow trout) in a limit test conducted at 100 mg/L.

Key value for chemical safety assessment

Additional information

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.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of approximately 14°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. Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 97% to 102% of nominal value and so the results are based on nominal test concentrations only. The 96-Hour LC50 based on nominal test concentrations was greater than 100 mg/L.

As there were no effects seen in fish up to the regulatory limit concentration of 100 mg/L, no key effect value for chemical safety assessment is relevant for fish and the substance is not considered to be hazardous to this aquatic trophic level.