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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
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.
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:100 mg/l was used (Limit Test)

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

Duplicate samples and samples at 48 and 72 hours were taken and stored frozen ( approximately -20°C) for further analysis if necessary.

The aqueous stock soluitions used to dose the dynamic, continuous flow apparatus were also taken at approximately -22, 0 and 72 hours for quantitative analysis. Duplicate samples were taken and stored frozen ( approximately -20°C) for further analysis if necessary.
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 nominal test concentrations of 1.0, 10 and 100 mg /l. The test item was dissolved directly in water.
An amount of test item (4.0 mg) was dissolved in dechlorinated tap water with the aid of utrasonification for approximately 5 minutes and the volume adjusted to 1.0 litre to give a 4.0 g/l stock solution. Aliquots (5.5, 55 and 550 ml) of the stock solution were each separately dispersed in dechlorinated tap water and the volume adjusted to 22 litres to give the 1.0, 10 and 100 mg/l test concentration respectively.The stock solution was inverted several times and the test concentration stirred with a flat blade stirrer to ensure they were fully mixed.
In the range-finding test 3 fish were added to each 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.
Given that the test material was suspected to decompose in solution when exposed to air, each test vessel was filled to 22 litres to reduce headspace and was covered and sealed


Definitive test:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/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 prepared by direct solution in water.
Information indicated that the test material may decompose in solution when exposed to air. Therefore the definitive study was conducted under dynamic, continuous flow testing conditions and the aqueous stock solutions were adjusted to pH 4.0 as determination of the physico-chemical properties of the test material indciated that oxidation was inhibited by lowering the pH to around 4.0

An amount of test item (12.2 mg) was dissolved in dechlorinated reverse osmosis water (adjusted to pH 4.0 with 10 M hydrochloric acid prior to the addition of test material) to 2 litres to give a 12.2 g/ 2 l stock solution. Stock solutions were freshly prepared in duplicate each day prior to dosing the dynamic, continuous flow apparatus to give a 100 mg/l test concentration. The stock soluitions were continuously stirred during the dosing by magenetic stirrer.

The concentration and stability of the test item in the test solutions were verified by chemical analysis at 0, 24 and 96 hours.

The aqueous stock solutions used to dose the dynamic, continuous flow system were also analysed at approximately -22, 0 and 72 hours.
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, UK. and maintained in house since 6 January 2000. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 2 February 2000 to 14 February 2000.


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 4.3 cm (sd =0.2) and a mean weight of 0.84 g (sd = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.42 g bodyweight/litre (static volume), 0.10 g of bodyweight/litre (volume in 24 hours) and 4.4 tank volumes per day.




Test type:
flow-through
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 150 mg/l as CaCO3.
Test temperature:
The test vessels were covered and maintained at approximately 14ºC.
pH:
The pH was measured daily.
See attached background information
Dissolved oxygen:
The dissolved oxygen concentration was measured daily
See attached background information
Nominal and measured concentrations:
FOR LIMIT TEST
Based on the results of the range-finding test a "Limit test" was conducted at a concentration (nominal) of 100 mg/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:
Range-finding test:
The range finder study was conducted under static test conditions, all test vessels were covered and sealed and a volume of 22 litres was used to minimise headspace and prevent losses of the test material due to oxiadation. At the start of the test three fish were placed in each test and control vessel and maintained at 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.

Definitive test:
Glass exposure vessels conatining 20 litres of diluent were used for each test concentration under dynamic, continuous flow testing conditions. The diluent supply was prepared at 60 ml/min by a Watson-Marlow 603s peristaltic pump with aqueous stock lines dosed by a Watson-Marlow 202 peristaltic pump at 1.0 ml/minute. The dosing apparatus was set up approximately 22 hours prior to the study start to allow equilibration of test concentration.
At the start of the study 10 fish were placed in each test vessel at random, in the prepared solutions. The test vessels were then covered to reduce evaporation and maintained at 14.0°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 received no auxillary aeration, the diluent supply only was aerated. 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 material


Physico-chemical measurements:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.
Oxygen concentrations were closely monitored during both studies. The results show higher oxygen concentrations during the definitive study, indicating that the use of a dynamic testing regime minimised oxygen depletion due to oxidation due to the continual replacement of the test media. The use of different test volumes and testing regimes for the range finding and definitive studies was considered not to affect the results or the validity of the study as toxicity was shown to be the same for both studies.
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
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
RANGE-FINDING TEST
There was no mortality to rainbow trout exposed to the test item at any concentrations (10, 10 or 100 mg/l) during the range-finding test. There were no sub-lethal effects of exposure during the range-finding test. See attached background information.

Based on this information, a single test concentration of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.


DEFINITIVE TEST
MORTALITY DATA:
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in the attached background information.
There were no mortalities in 20 fish exposed to a test concentration of 100 mg/L for a period of 96 hours. Inspection of the mortality data gave the following results:
3 h LC50: >100 mg/L
6 h LC50: >100 mg/L
24 h LC50: >100 mg/L
48 h LC50: >100 mg/L
72 h LC50: >100 mg/L
96 h LC50: >100 mg/L

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg/L, the lowest test concentration resulting in 100% mortality to be greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

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

SUB-LETHAL EFFECTS:
There were no sub-lethal effects of exposure observed in 20 fish exposed to a test concentration of 100 mg/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 (see attached background material). Temperature was maintained at approximately 14°C throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependednt differences in pH were observed throughout the study. The pH of the 100 mg/l replicates R1 and R2 were obseved to be between 1.0 and 1.2 pH units lower than the control vessel throughout the study. This was considered to be due to the lowered pH of the stock solutions used to dose the dynamic continuous flow test system.
It was considered necesasry to lower the pH of the stock solution to prevent oxidation of the test material. The pH of the test media during the study was within the recommendations of the test guidelines and the test fish showed no adverese reactions to the lower pH. Therefore the test guidelines and the test fish showed no adveres reactions to the lowered pH. Therefore this effect was considered not to adverseley effect the results or the valididty of the study.

VERIFICATION OF TEST CONCENTRATIONS:
Pre-study recovery and stability anaylses conducted and information indicated the test material to be highly unstable in the test diluent. This was considered to be due to possible oxidation of the test. material. Therefore the definitive study was conducted under dynamic, continuous flow testing conditions in the attempt to maintain near nominal test concentrations. The aqueous stock solutions used to dose this test system were adjusted to a pH of approximately 4.0 as the determination of the general physico-chemical properties of the test material showed reduced rate of oxidation at lowered pH.
Given that the test material was suspected to be unstable, analysis was performed for the parent test material and a known oxidation product in the aqueous stock solutions and the test media during the definitive study. Analysis of both the aqueous stock solutions and the test media showed measured concentrations of the parent test material ranging from 85% to 111% of nominal values, whilst measured concentrations of the oxidation product in the test samples were very low with values of 1.33mg/l and 4.75 mg/l.


VALIDITY CRITERIA:
There were no mortalities or signs of stress in the control fish during the 96 hours.
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 >100 mg/l. The No Observed Effect Concentration was 100 mg/l.
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 Directive 92/69/EEC.

Methods

Following a preliminary range-finding test fish were exposed, in two groups of ten, 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 dynamic 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

The 96-Hour LC50based on nominal test concentrations was > 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.

Pre-study recovery and stability anaylses conducted and information indicated the test material to be highly unstable in the test diluent. This was considered to be due to possible oxidation of the test. material. Therefore the definitive study was conducted under dynamic, continuous flow testing conditions in the attempt to maintain near nominal test concentrations. The aqueous stock solutions used to dose this test system were adjusted to a pH of approximately 4.0 as the determination of the general physico-chemical properties of the test material showed reduced rate of oxidation at lowered pH.

Given that the test material was suspected to be unstable, analysis was performed for the parent test material and a known oxidation product in the aqueous stock solutions and the test media during the definitive study. Analysis of both the aqueous stock solutions and the test media showed measured concentrations of the parent test material ranging from 85% to 111% of nominal values, whilst measured concentrations of the oxidation product in the test samples were very low with values of 1.33mg/l to 4.75 mg/l.

Given that toxicity cannot be attributed to the parent test material or the oxidation product but to a combination of them all, and as the measured concentrations of the parent test material were all shown to be in excess of 80% nominal the LC50 values are based on the nominal test concentrations only.

Description of key information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 >100 mg/l.  The No Observed Effect Concentration was 100 mg/l.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

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 Directive 92/69/EEC.

Methods

Following a preliminary range-finding test fish were exposed, in two groups of ten, 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 dynamic 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

The 96-Hour LC50based on nominal test concentrations was > 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.

Pre-study recovery and stability anaylses conducted and information indicated the test material to be highly unstable in the test diluent. This was considered to be due to possible oxidation of the test. material. Therefore the definitive study was conducted under dynamic, continuous flow testing conditions in the attempt to maintain near nominal test concentrations. The aqueous stock solutions used to dose this test system were adjusted to a pH of approximately 4.0 as the determination of the general physico-chemical properties of the test material showed reduced rate of oxidation at lowered pH.

Given that the test material was suspected to be unstable, analysis was performed for the parent test material and a known oxidation product in the aqueous stock solutions and the test media during the definitive study. Analysis of both the aqueous stock solutions and the test media showed measured concentrations of the parent test material ranging from 85% to 111% of nominal values, whilst measured concentrations of the oxidation product in the test samples were very low with values of 1.33mg/l to 4.75 mg/l.

Given that toxicity cannot be attributed to the parent test material or the oxidation product but to a combination of them all, and as the measured concentrations of the parent test material were all shown to be in excess of 80% nominal the LC50 values are based on the nominal test concentrations only.