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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:
13 September to 15 October 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP using OECD method 203. Purity of TBTC 96.1%. Study used water accomadated fractions which are not intended for organometallic substances.
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:
Dilutions containing 0.010, 0.032, 0.10, 0.32 and 1.0 % of a WAF prepared at a loading rate of 100 mg/L.

- Sampling method:
During the final test triplicate samples for analysis were taken from all test concentrations including the control. In addition, samples were daily taken from the freshly prepared undiluted WAF.

Sampling:
Frequency - at t=0 h and t=72 h from freshly prepared solutions and at t=24 h and t=96 h from 24-hour old solutions.
Volume - 11 mL from the approximate centre of the test vessels.
Storage - Samples were stored at room temperature for a maximum of 7 days until transportation to TNO Nutrition and Food Research.

Additionally, singular reserve samples of 11 mL were taken for possible analysis according to the above schedule.

- Sample storage conditions before analysis:
If not already used, these samples were stored at room temperature for possible analysis for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The standard test procedures required generation of test solutions that contain completely dissolved test material concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturbed the test system were prevented (e.g. film of the test material on the water surface).

The batch of test material tested was a colourless liquid with a reported purity of 96.1 %. It is considered insoluble in water (solubility <0.1 mg/L at 20 °C).

Preparation of test solutions started with dispersions of the stock solution in test medium at a nominal loading of 100 mg/L. The dispersions were stirred for approximately 24 hours applying an ultra thurrax, after which they were left to stabilise for approximately 24 hours. Subsequently, the water fractions were separated from the undissolved fraction of test material by means of siphoning into a separation funnel. After another short stabilisation period in the separation funnel, the Water Accommodated Fraction (WAF) was collected for use in the test. The lower test concentrations were prepared by subsequent dilution of the WAF in test medium. All test vessels were pre-conditioned for approximately 30 minutes with 1 litre of the corresponding test solutions prior to the start of the test. Test solutions were renewed daily according to the above procedure.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra-fish
- Strain: Brachydanio rerio, Teleostei, Cyprinidae
- Age at study initiation (mean and range, SD): No data
- Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.
- Length at study initiation (length definition, mean, range and SD): 3.2 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.63 ± 0.16 g
- Feeding during test: No feeding from 48 hours prior to the test and during the total test period.

ACCLIMATION
- Acclimation period: At least 12 days after delivery.
- Health during acclimation (any mortality observed): Healthy fish supplied with a health certificate.
- Type and amount of food: Daily with Trouvit
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
n/a
Hardness:
After aeration the hardness was 250 mg CaCO3 per litre.
Test temperature:
21-25 °C constant within ± 1 °C
pH:
6.0-8.5
Dissolved oxygen:
oxygen > 60 % of air saturation value
Salinity:
n/a
Nominal and measured concentrations:
Dilutions containing 0.010, 0.032, 0.10, 0.32 and 1.0 % of a WAF prepared at a loading rate of 100 mg/L.

Mean measured concentrations: < 1.3, < 1.3, 3.9, 16 and 42 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 litres, all glass
- Aeration: The test media were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): Semi-static with daily renewal of test solutions.
- No. of organisms per vessel: 7
- Biomass loading rate: 0.49 g fish/litre, i.e. 7 fish per 9 litres of test medium.


TEST MEDIUM / WATER PARAMETERS
ISO-medium [ISO International Standard 7346], aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilised. After aeration the hardness was 250 mg CaCO3 per litre and the pH was 8.0.

ISO-medium [ISO International standard 7346]: the following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water (tap water) purified by reverse osmosis (milli-RO); Millipore Corp., Bedford, Mass., USA)
(mg/1): CaCl2.2H20 293.8
MgSO4.7H20 123.3
NaHCO3 64.8
KCl 5.8
- Dissolved oxygen content, pH and temperature were measured daily in all vessels, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily
- Introduction of fish: Within half an hour after preparation of the test media.
- Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2 % ethylene glycol monophenylether in water.

EFFECT PARAMETERS MEASURED
The objectives of the study were to determine:
• The 3, 24, 48, 72 and 96 h LC50 of the test material, i.e., the concentration killing 50 % of the exposed fish species Brachydanio rerio (zebra-fish) at the given time under experimental conditions. Mortality and other effects were therefore measured at 3, 24, 48, 72 and 96 hours following the start of exposure.
• The 96 h minimum concentration of the test substance producing total mortality, i.e., 96h-LC100.
• The 96 h maximum concentration of the test substance producing no mortality, i.e., 96h-LC0.
• The 96 h No Observed Effect Concentration (NOEC) of the test substance for the fish species with respect to mortality and with respect to condition.

The validity of the test results is based on the following criteria:
- The proportion of control fish showing abnormal behaviour (including mortality) will not exceed 10 % (or one fish if less then ten are used) by the end of the test.
- The oxygen concentration will be maintained at at least 60 % of the air saturation value throughout the test (> 5 mg/L at 22 °C). Other test conditions (pH and temperature) will be maintained within the limits prescribed by this protocol.
- An analytical program will be included to provide for evidence that the actual test concentrations have been maintained between 80 and 120 % of the initial concentrations.
- The 96h-LC50 of the reference chemical, if available, for the stock of fish was in reasonable agreement with results obtained previously in the same laboratory.


TEST CONCENTRATIONS
- Range finding study
Combined limit/range-finding test:
The project started with a combined limit/range-finding test. Test procedure and conditions were similar to those applied in the final test with the following exceptions: 7 fish per concentration were exposed to a blank-control and a WAF prepared at a loading rate of 100 mg/L, while three fish per concentration were exposed to dilutions containing 0.1, 1.0 and 10 % of the WAF. Dissolved oxygen concentrations, temperature and pH were only measured in the control and the highest test concentration with surviving fish. Triplicate samples for possible analysis were taken from the WAF prepared at 100 mg/L and the ten-fold dilution (10 % WAF). In addition, singular reserve samples were taken from these two solutions.


Additional range finding test:
The project was continued with an additional range-finding test. Test procedure and conditions were similar to those applied in the final test with the following exceptions: three fish per concentration were exposed to dilutions containing 0.001, 0.01 and 0.1 % of a WAF prepared at a loading rate of 100 mg/L. Dissolved oxygen concentrations and pH were, at least, measured in the lowest and the highest test concentration. The range-finding test did not include sampling for determination of actual concentrations.

- Results used to determine the conditions for the definitive study:
Combined limit/range-finding test:
All fish exposed to the various TBTC treated test groups died within 72 hours after exposure. It should be noted that fish exposed to the undiluted WAF already died within 10 minutes of exposure. Two fish exposed to the test group representing 0.1 % of the WAF were euthanised for humane reasons after 72 hours as they showed haemorrhage and were immobilised and expected to die shortly after this observation. The study was stopped.

The measured TBTC concentration at the start of exposure in the undiluted WAF was 10.3 mg/L (mean of triplicate measurement). The concentration remained stable during the first 24-hour renewal period. The mean concentration measured in the sample containing 10 % of the WAF was 1.0 mg/L and thus in agreement with the dilution factor of ten. This concentration also remained stable during the first 24-hour test period.


Additional range-finding test:
No effects were observed in any of the WAF dilutions tested. The differences observed between the combined limit/range-finding test and the additional range-finding test at 0.1 % WAF were considered to be related to differences in actual exposure concentration. Based on all test results available it was decided to continue with a final LC50 test with test groups representing a range between 0.01 and 1.0 % of a WAF prepared at a loading rate of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
pentachlorophenol
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.9 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.9 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Hypoactive swimming
- The results of the final test are presented in Table 4. Table 5 specifies the clinical effects observed at the different test groups. No mortality or visible clinical effects were observed in the dilutions containing 0.010, 0.032 and 0.10 % of the WAF prepared at 100 mg/L during the 96-hour test period. All fish exposed to the dilutions containing 0.32 and 1.0 % of the WAF died during the test period.


- The results of measurement of pH and oxygen concentrations are presented in Tables 6 and 7. The temperatures measured during the study in the various test vessels are presented in Table 8. All test conditions remained within the ranges prescribed by the protocol (pH: 6.0-8.5, constant within 1 unit; temperature 21 -25 °C, constant within ± 1 °C; oxygen > 60 % of air saturation value).
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Mortality/LC50:
Under the conditions of the present test with zebra-fish exposed to PCP, the 96h-LC50 was estimated to correspond to 0.11 mg/L based on nominal concentrations, with 20 % mortality at 0.10 mg/L and 100 % mortality at 0.22 mg/L. This effect was already reached within 48 hours of exposure.
- The range of the 96h-LC50 for zebra-fish is generally between 0.10 mg/L and 0.46 mg/L based on historical data of reference tests performed by NOTOX. Hence, the sensitivity of zebra-fish originating from the present batch for PCP is relatively high in comparison to the sensitivity generally observed during the past years.
Reported statistics and error estimates:
Treatment of the results:
LC-values: The 48h-LC50 was determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations
The LC50 at 24 and 72 hours following exposure could not be determined using the maximum likelihood estimation method. This was because there was no concentration between the highest concentration (A) at which 0 % mortality and the lowest concentration (B) at which 100 % mortality occurred. Instead, the LC50 was calculated as (AB)½, with A and B being limits of the 95 % confidence interval.
NOEC values: The No Observed Effect Concentrations (NOEC values) are the highest concentrations tested showing no effect throughout the exposure time. The NOEC values are qualified according to the duration of exposure and are estimated by comparing effects with regard to survival of the exposed animals with those of the control animals.

Measured concentrations:

The measured test material concentrations in the various WAF dilutions, with detectable concentrations, decreased slightly during the 24-hour renewal periods. Analyses showed acceptable repeatability of the WAF preparation with measured test material concentrations in the freshly prepared solutions between 5900 and 8300 µg/L in the undiluted WAF prepared at 100 mg/L. An overview of the mean concentrations measured including the average exposure concentrations are presented in Table 3.

Acceptability of the test:

- In the control group, none of the fish showed abnormal behaviour (including mortality).

- The oxygen concentration was maintained at at least 60 % of the air saturation value throughout the test (> 5 mg/L at 22 °C). Further, the other test conditions (pH and temperature) were maintained within the limits prescribed by this protocol.

- The analytical program showed that the actual concentration had not been maintained at more than 80 % of the initial concentration during the two sampled 24-hour renewal periods. However, the method of preparation and collecting the maximum soluble fraction from the freshly prepared WAF was the most optimal. The differences in the freshly prepared concentrations and the following decrease of concentration could not be prevented, as these were a result of the behaviour and extremely low solubility of the test substance. Consequently, average exposure concentrations were calculated.

- The 96h-LC50 of the reference chemical for the stock of fish was in agreement with results obtained previously at NOTOX.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present test the test material induced no mortality or visible effects in zebra-fish (Brachydanio rerio) at or below a concentration containing 0.10 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L. Based on average measured concentrations the NOEC corresponded to 3.9 µg/L.
The 96h-LC50 for zebra-fish exposed to the test material equalled a concentration containing 0.57 % of a WAF prepared at a loading rate of 100 mg/L. Based on average measured concentrations the 96h-LC50 corresponded to 7.9 µg/L.
Executive summary:

The 96 -Hour Acute Toxicity of the test material in zebra-fish was investigated. The study procedures described in this report were based on the ISO International Standard 7346-2: Semi-static method, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992 and the OECD guideline No. 203, 1992. The batch of the test material was a colourless liquid with a reported purity of 96.1 %. The test material is considered insoluble in water (solubility <0.1 mg/L at 20 °C).

Preparation started with a supersaturated dispersion at 100 mg/L, which was magnetically stirred for approximately 24 hours. The dispersion was then left to stabilise for 24 hours. Subsequently, the Water Accommodated Fraction (WAF) was separated from the undissolved fraction of test material by means of siphoning. The lower test concentrations were prepared by subsequent dilution of the WAF in test medium. All test solutions were renewed daily and all test vessels were pre-conditioned for 30 minutes with the respective solution.

The project started with a combined limit/range-finding test in a semi-static system with daily renewal. Seven zebra-fish per test group were exposed to a blank control and a WAF prepared at a test material loading rate of 100 mg/L in the limit test, while three zebra-fish per test group were exposed to dilutions containing 0.1, 1.0 and 10 % of the WAF in the additional range-finding test. The results showed that all fish exposed to the various test material treated test groups died within 72 hours of exposure. Analyses of samples taken from the test groups containing the undiluted WAF and the group containing 10 % of the WAF showed that measured concentrations were stable during the first 24-hour renewal period. Concentrations measured were approximately 10 mg/L in the undiluted WAF and 1.0 mg/L in the ten-fold dilution of the WAF (10 %).

The project was continued with an additional range-finding test. Three fish per concentration were exposed to dilutions containing 0.001, 0.01 and 0.1 % of a WAF prepared at a loading rate of 100 mg/L. No effects were observed in any of the WAF dilutions tested. The differences observed between the combined limit/range-finding test and the additional range-finding test at 0.1 % WAF were considered to be related to differences in actual exposure concentration.

Based on all test results available it was decided to continue with a final LC50 test with test groups representing a range between 0.01 and 1.0 % of a WAF prepared at a loading rate of 100 mg/L. Seven fish were exposed per test group and a blank control in a semi-static system with daily renewal.

Analysis during the final test showed that the measured test material concentrations in the various WAF dilutions, with detectable concentrations, decreased slightly during the 24-hour renewal periods. Analyses showed acceptable repeatability of the WAF preparation with measured test material concentrations in the freshly prepared solutions between 5900 and 8300 µg/L in the undiluted WAF prepared at 100 mg/L.

The average exposure concentrations for the dilutions essential for the determination of the toxicity parameters were 3.9, 16 and 42 µg/L at the corresponding WAF dilutions containing 0.10, 0.32 and 1.0 % of the WAF, respectively.

The study met the acceptability criteria prescribed by the protocol and the guidelines and was considered valid.

The test material induced no mortality or visible effects in zebra-fish (Brachydanio rerio) at or below a concentration containing 0.10 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L in the final test.

The 96h-LC50 for zebra-fish exposed to the test material equalled a concentration containing 0.57 % of a WAF prepared at a loading rate of 100 mg/L. Based on average measured concentrations the 96h-LC50 corresponded to 7.9 µg/L. The 96h-LC50 was already reached after 72 hours of exposure.

Description of key information

A 96-Hour Acute Toxicity Study in zebra-fish was conducted with the test material. The study procedures described in this report were based on the ISO International Standard 7346-2: Semi-static method, 1996.

The average exposure concentrations for the dilutions essential for the determination of the toxicity parameters were 3.9, 16 and 42 µg/L at the corresponding WAF dilutions containing 0.10, 0.32 and 1.0 % of the WAF, respectively.

The study met the acceptability criteria prescribed by the protocol and the guidelines and was considered valid.

The test material induced no mortality or visible effects in zebra-fish (Brachydanio rerio) at or below a concentration containing 0.10 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L in the final test.

The 96h-LC50 for zebra-fish exposed to the test material equalled a concentration containing 0.57 % of a WAF prepared at a loading rate of 100 mg/L. Based on average measured concentrations the 96 h-LC50 corresponded to 7.9 µg/L. The 96h-LC50 was already reached after 72 hours of exposure.

Key value for chemical safety assessment

LC50 for freshwater fish:
7.9 µg/L

Additional information

In the key study (Migchielsen, 2004) a 96-Hour Acute Toxicity Study in zebra-fish was conducted with the test material. The study procedures described in this report were based on the ISO International Standard 7346-2: Semi-static method, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992 and the OECD guideline No. 203, 1992. The batch of test material tested was a colourless liquid with a reported purity of 96.1 %. The test material is considered insoluble in water (solubility <0.1 mg/l at 20 °C) and so a WAF method was used.

The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as the study used water accomadated fractions which are not intended for organometallic substances. This study was conducted most recently and had less deviations than the supporting studies that are available and was therefore chosen as the most relevant and reliable study.

The following supporting studies are also available and correlate with the key study:

- Supporting information is available from a test material safety data sheet, Atofina Chemicals Inc. (2001).

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source. The LC50 for the test material on fish in freshwater was reported to be 0.02-0.021 mg/L.

- Supporting information is also available from another test material safety data sheet, Atofina Chemicals Inc. (2001).

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source. The LC50 for the test material on fish in brackish water was reported to be 0.026-0.045 mg/L.

- In the Bushong et al (1987) paper, Chesapeake Bay fish species were exposed to a range of tributyltin (TBT) concentrations using continuous-flow conditions. TBT measurements were reported in the test chambers every 24 h; test solutions were generally within 10-15 % of the predicted concentrations. The most acutely sensitive fish species tested were larval inland silversides (Menidia beryllina), (72 h LC50 = 4.6 µg/L TBT) and juvenile Atlantic menhaden (Brevoortia tyrannus), (96 h LC50 = 4.5 µg/L TBT). M. Medinia had an LC50 of 8.9 µg/L. These experiments demonstrate that TBT concentrations of <1.0 µg/L and <5.0 µg/L can be acutely toxic to some fish species. A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as there was limited information on test material and no information on GLP however methodolgy is well documented and also non-standard fish species were used.

- In the Bushong et al (1988) paper, acute tributyltin (TBT) toxicity experiments were conducted on selected Chesapeake Bay biota. Five fish species were evaluated. Tests were conducted using continuous-flow conditions and TBT concentrations were measured every 24 h in test containers. Test solutions were generally within 10-15 % of the predicted concentrations.

The most sensitive fish species tested were larval inland silverside (Menidia Beryllina), (96-h LC50 = 3.0 µg L-1 TBT) and juvenile Atlantic menhaden (Brevoortia tyrannus), (96-h LC50 = 4.5 µg L-1 TBT). Mummichogs (Fundulus heteroclitus) and sheepshead minnow (Cyprinodon variegatus), (96 h LC50 = 23.8 and 25.9 µg L-1 TB). respectively) were the most resistant fish species tested. A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as there was limited information on test material and no information on GLP however methodolgy is well documented and also non-standard fish species were used.

- In the Nagase et al (1991) paper, the study was conducted according to the OECD 403 method, however there was no information given on GLP and purity therefore a reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997. The LC50 for the test material is 1.11 x 10-4mmol dm-3(0.036 mg/L).

- In the Seinen et al (1981) paper, Rainbow trout in the yolk sac fry stage were continuously exposed to the test material at concentrations of 0, 0.2, 1 and 5ppb (µg/L) for 110 days. After a 10 to 12 days exposure period, at the transition of the yolk sac fry stage to the swimming fry stage, all fish of the 5 ppb group suddenly died without any preceding symptoms. At these exposure levels the test material induced a significant and dose related growth retardation resulting in a 44 % decrease of the body weights in the 1 ppb group at the end of the experimental period. The 10-12 day LC100 was considered to be 0.005 mg/L

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as no guideline was specified, however methodolgy is well documented.