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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-25 to 2012-05-18
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:
Range-finding studyStability - A sample of each loading rate WAF was taken for analysis at 0 and 24 hours. All samples were stored at approximately -20°C prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.Definitive testConcentration and stability - Samples take at 0 (fresh media), 24 and 96 hours (old media)Water samples taken from control and 100 mg/L laoding rate WAF test group at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. All samples were stored at approximately -20°C prior to analysis.Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (old and fresh media), were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Validation of mixing periodPre-study investigational work was performed to determine if stirring for a prolonged period produced significantly higher levels of total organic carbon (TOC), as an indicator of soluble organic substances, inthe WAF.A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for 95 hours. After a 1-hour standing period, the mixtures were removed by siphon and samples taken for TOC.Range-finderAmounts of test item (210 and 2100 mg) were each seperately added to the surface of 21 litres of dechlorinated tap water to give 10 and 100 mg/L loadingrates respectively. After the addition of the test item the water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped aafter 23 hours andthe mixtures allowed to stand for 1 hour. Microscopic observations of made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was justified to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 - 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations were performed onthe WAFs after filtering and showed no micro-particles of the test item present.Definitive testA limit test at a single loading rate of 100 mg/L was conducted to confirm the range-finder results.The test concentration was prepared in the same manner as for the range finder.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM- Common name: Rainbow trout- Source: Brow Well Fisheries Ltd. Hebden, nr Skipton, Yorkshire, UK- Age at study initiation (mean and range, SD): not stated. maintained in-house since 25 April 2012- Length at study initiation (length definition, mean, range and SD): 4.4 cm (s.d. = 0.4)- Weight at study initiation (mean and range, SD): 0.68 g (s.d. = 0.23)- Feeding during test: no discontinued 24 hours prior to start of definitive test.- Food type: commercial trout pelletsACCLIMATION- Acclimation period: 26 April 2012 - 08 May 2012- Acclimation conditions (same as test or not): Yes- Type and amount of food: commercial trout pellets- Health during acclimation (any mortality observed): < 1% mortality
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
140 mg/L as CaCO3
Test temperature:
14 - 15°C
pH:
7.7 - 8.3
Dissolved oxygen:
9.5 - 10.2 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
Range finder: 10 and 100 mg/L loading rate WAFDefinitive: 100 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM- Test vessel: 20 L glass exposure vessel- Type: closed- Aeration: yes- Renewal rate of test solution (frequency/flow rate): single pass- No. of organisms per vessel: 7- No. of vessels per concentration (replicates): 1- Biomass loading rate: 0.24 g bw/LTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Water Softner).- Total organic carbon: average = 0.920 mg/L- Pesticides:0.009 µg/L- Chlorine: 0.27 mg/L- Conductivity: 419 µSi/cm at 20°C- Culture medium different from test medium: NoOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: 16 hours light/8 hours darkness with 20 minute dawn and dusk transition periodsEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Mortality and sub-lethal effects: 24, 48, 72 and 96 hours after exposure.TEST CONCENTRATIONS- Range finding study- Test concentrations: 10 and 100 mg/L loading rate WAF- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Loading Rate Water Accomodated Fraction (WAF)
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Loading Rate Water Accomodated Fraction (WAF)
Details on results:
There were no mortalities in 7 fish exposed to a 100 mg/L laoding rate WAF for a period of 96 hours.The results of the definitive test showed the highest loading rate resulting in 0% mortality to be greater than or equal to 100 mg/L loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.There were no sub-lethal effects of exposure observed in 7 fish exposed to 100 mg/L loading rate WAF for a period of 96 hours.The oxygen concentration in some test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.At the start and end of each mixing period, and after the 1-hour settlement period the 100 mg/L loading rate was observed to be a clear colourless water column with oily globules or test item at the water surface and dispersed throughout the water column. Visual examination of the WAF showed globules of test item to be present in the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug showed no microscopic particles of test item to be present. During the test the 100 mg/L loading rate was observed to be a clear colourless solution.Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method , the results were based on nominal loading rates only.
Results with reference substance (positive control):
Not applicable

See attached document for analytical results.

Validity criteria fulfilled:
yes
Conclusions:
The 96 h LL50 based on nominal loading rates was greater than or equal to 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.
Executive summary:

Test Guidance

OECD Guideline No. 203 and EC Method C.1

Method

Following a prelliminary range-finding test, seen fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of approximately 14°C under semi-static 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.

Result

The 96 h LL50 based on nominal loading rates was greater than or equal to 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of test preparations at 0 (fresh media, 24 and 96 hours (old media) showed measured concentrations to range from less than the limit of quantification (LOQ) of the analytical method (assessed as 0.34 mg/L) to 0.547 mg/L.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002-05-26 to 2002-07-05
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:
no
Details on sampling:
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 was measured using a WTW pH 320 pH meter and the dissolved oxygen concentration was measured using a YSI 550 dissolved oxygen meter at 0 to 72 hours and a YSI 57 dissolved oxygen meter at 96 hours.
Details on test solutions:
Range-finderAmounts of test material (210 and 2100 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed tostand for 1 hour. A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material, therefore a glass wool plug was not used to filter the WAFs.Definitive testBased on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L. The required amount of test material was added to each test vessel using the method described for th erange-finder.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncovhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near. Skipton, Yorkshire, UK and maintained in-house since 29 May 2002. Fish were maintained in a glass fibre tank with a "single pass" water renewal system.The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.6 cm (sd = 0.4) and a mean weight of 1.30 g (sd = 0.33) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.65 g bodyweight/L.The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
100 mg CaCO3/L
Test temperature:
14°C
pH:
7.5 - 7.9
Dissolved oxygen:
9.2 - 9.9 mg O2/L
Salinity:
Not appliable
Nominal and measured concentrations:
range finder: 10 amd 100 mg/LDefinitive test: 100 mg/L
Details on test conditions:
TEST SYSTEMrange finder:In the range-finding test 3 fish were added to each 20 litre test and control vessel 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 under static test conditions.The control group was maintained under identical conditions but not exposed to the test material.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.Definitive testAs in the range-finding test 20 litre glass exposure vessels were used for each test concentration.At the start of the test 10 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 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 were aerated via narrow bore glass tubes. The fish were not individually identified and received no food duringexposure.The control group was maintained under identical conditions but not exposed to the test material.A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure test concentrations of soluble components of the test material were maintained over the test 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.TEST MEDIUM / WATER PARAMETERSLaboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve therequired temperature.- Total organic carbon: 1.0 mg/L- Pesticides: < 0.01 µg/L- Chlorine: (Total: 0.34 mg/L)- Alkalinity: 102 mg/L- Conductivity: 558 µS/cmOTHER TEST CONDITIONS- Adjustment of pH: noTEST CONCENTRATIONS- Range finding study- Test concentrations: 10 and 100 mg/L- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: loading rate WAF
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: loading rate WAF
Details on results:
Range finderThere were no sub-lethal effects of exposure during the range-finding test.The results showed no mortalities at the 10 and 100 mg/L loading rate WAFS. Based on this information, a single loading rate, in duplicate, of 100 mg/L using a stirring period of 24 hours followed by a l-Hour standing period was selected for the definitive test. Definitive testThe EL50 was > 100 mg/L loading rate WAF at all timepoints in the study.The results of the definitive test showed the highest loading rate resulting in 0% mortality to be greater than or equal to 100 mg/L loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 100 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate.There were no sub-lethal effects of exposure observed in 20 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.Temperature was maintained at 14.0°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.The pH of the control group was observed to vary between 7.5 and7.9. This variation was considered not to affect the validity or integrity of the study given that no mortalities or adverse reactions to exposure were observed in the control group and the Test Guideline states that the pH should not vary by more than 1 unit.Observations on the test media were carried out during the mixing and testing of the WAFS. At the start and end of each mixing period, and after the 1-Hour settlement period the 100 mg/L loading rate was observed to be a clear colourless water column with white opaque globules at the surface and on the bottom of the vessel. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colourless solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present, therefore a glass wool plug was not used to filter the WAF.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
none
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.
Executive summary:

Test Guideline

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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 (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following a preliminary range-finding test fish were exposed, in two groups of ten, to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14.0°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.

The 96-Hour LL50 based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

The 96 h LL50 based on nominal loading rates was greater than or equal to 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L (OECD 203 and EU Method C.1).

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

Supporting study

Test Guideline

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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 (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following a preliminary range-finding test fish were exposed, in two groups of ten, to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14.0°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.

The 96-Hour LL50 based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Key study

Test Guidance

OECD Guideline No. 203 and EC Method C.1

Method

Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of approximately 14°C under semi-static 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.

Result

The 96 h LL50 based on nominal loading rates was greater than or equal to 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of test preparations at 0 (fresh media, 24 and 96 hours (old media) showed measured concentrations to range from less than the limit of quantification (LOQ) of the analytical method (assessed as 0.34 mg/L) to 0.547 mg/L.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.