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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:
25 January 2003 - 31 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guidelines for Testing of Chemicals (1992) No. 203, and United Kingdom (UK) Good Laboratory Practice (GLP)
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
A volume of test material was diluted with methanol (1:1) and analysed.

Standard solutions of test material were prepared in methanol/water (50:50 v/v) at a nominal concentration of 10 mg/l.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and high purity of the test material the test concentrations used in the definitive test were prepared by diluting a saturated solution prepared from initial test material dispersions at a concentration of 150 mg/L.
An amount of test material (3.375g) was dispersed in 22.5 liters of dechlorinated tap water with the aid of propeller stirring at approximately 2000 rpm at 25 degC for a period of 48 hours. After 48 hours the stirring was stopped, the mixtures cooled to 14 degC and the undissolved test material removed by filtration (0.2 microm Gelman SuporCap filter, first approximate 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 12 mg/L. The nominal 12 mg/L test concentration was prepared in triplicate to ensure a sufficient volume was available for testing and dilution. Aliquots (1.117, 2, 3.50, 6.333 and 11.17 liters) of this nominal 12 mg/L test concentration were each separately dispersed into a final volume of 20 liters of dechlorinated tap water and stirred using a flat bladed mixer for approximately 1 minute to give the remainder of the test series of nominal test concentrations of 0.67, 1.2, 2.1, 3.8 and 6.7 mg/L.
Media preparation trials conducted using reverse osmosis purified water as the diluent indicated that when using a similar method of preparation as described above, a measured concentration (based on Dissolved Organic Carbon analysis) of a similar magnitude to the results of the water solubility test was obtained. The Determination of General Physical-chemical Properties test (4.8 Water Solubility, Safepharm, 2003) gave a water solubility value of 3.2 mg/L. It was therefore considered that filtration was an appropriate method for the removal of undissolved test material.

- Media preparation trials
Media preparation trials were conducted in order to determine the most suitable method of obtaining a saturated solution at a concentration similar to the water solubility level of 32 mg/L (Section 4.8 Safepharm, 2003).
Method 1:
An amount of test material (1650 mg) was dispersed in 11 liters of reverse osmosis purified deionized water with the aid of propeller stirring at approximately 2000 rpm for 48 hours to give an initial test material dispersion of 150 mg/L. After 48 hours stirring was stopped and the undissolved test material was removed by filtration (0.2 mircom Gelman Acrocap filter) to give a saturated solution. Samples were taken from this saturated solution to determine the amount of dissolved test material by Dissolved Organic Carbon (DOC) analysis. The amount of test material in solution was shown to be 23 mg/L.

Method 2:
An amount of test material (75 mg) was dispersed in 500 ml of reverse osmosis purified deionized water with the aid of shaking (INFORS Aerotron) at approximately 300 rpm at a temperature of 30 deg C for 48 hours. After 48 hours the shaking was stopped and the undissolved test material was removed by filtration (0.2 microm Gelman Acrocap filter) to give a saturated solution. Samples were taken from this saturated solution to determine the amount of dissolved test material by Dissolved Organic Carbon (DOC) analysis. The amount of test material in solution was shown to be 21 mg/L.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 15 January to 27 January 2003. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14 degC with dissolved oxygen content of greater than or equal to 9.6 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 48 hours prior to the start of the definitive test. Food was withheld for approximately 48 hours as opposed to 24 hours. However this was considered not to affect the results or validity of the study given that no mortalities or sub-lethal effects of exposure were observed in the control fish during the 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 4.2cm (sd = 0.2) and a mean weight 0.96g (sd = 0.18) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.48g bodyweight/liter.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Approximately 100 mg/l as CaCO3.
Test temperature:
Maintained at 13.0 to 14.4 deg C throughout the study.
pH:
Mean value of 7.804.
Nominal and measured concentrations:
0.67, 1.2, 2.1, 3.8 and 6.7 mg/l.
Details on test conditions:
- Range finding test
The results of the media preparation trials showed that the dissolved test material concentration obtained from both Methods 1 and 2 was slightly lower than the water solubility level of the test material as determined by 4.8 Water Solubility (Safepharm, 2002). This was considered possibly to be due to the filters used in the media preparation trials not been pre-conditioned by passing a suitable volume through the filter prior to taking the sample for DOC analysis. However, given that the results of the DOC analyses were of a similar magnitude to the results of the water solubility test, it was considered that filtration was appropriate for removal of the undissolved test material if pre-conditioning of the filters was conducted.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. For the purpose of the range-finding test the propeller stirring method was used for the preparation of a saturated solution of the test material,

An amount of test material (3375 mg) was dispersed in 22.5 liters of dechlorinated tap water with the aid of propeller stirring at approximately 2000 rpm at 25 degC for a period of 48 hours. After 48 hours the stirring was stopped, the mixture cooled to 14 degC and the undissolved test material removed by filtration (0.2 microm Gelman SuporCap filter, first approximate 1 liter discarded in order to pre-condition the filter) to give a saturated solution.
A saturated solution prepared in an identical manner to that used for the range-finding test to provide samples for preliminary recovery and stability analyses showed that the dissolved test material concentration in the saturated solution was 12 mg/l. This value was lower than the test concentration of 23 mg/l determined by DOC analysis during the media preparation trials when reverse osmosuis puridied deionised water was used as the diluent.
Examination of the test material structure indicated that it was a zinc salt with the anion being a weak acid. it was therefore considered that the weak acid may associate with other cations of limited solubility leading to precipitation. As a result of this the maximum solubility of the test material in dechlorinated tap water was reduced by the presence of cations. These cations were not present in the reverse osmosis purified water solubility test, hence higher dissolved test material concentrations were obtained in these diluents.
Based on the results of the recovery analyses conducted, it was considered appropriate to base the concentrations employed in the range-finding test on a nominal saturated solution concentration of 12 mg/l. Aliquots (190 ml and 1.9 liters) of this nominal 12 mg/l test concentration were each separately dispersed in a final volume of 20 liters of dechlorinated tap water and stirred using a flat bladed mixer for approximately 1 minute to give the remainder of the test series of 0.12 and 1.2 mg/l.
In the range-finding test 3 fish were added to each 20 litre test and control vessel and maintained at approximately 14 deg 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, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. Mortalities and sub-lethal effects of exposure were not determined after 6 hours exposure due to a technical oversight.

- Definitive test
Based on the results of the range-finding test and the preliminary recovery analyses conducted the test material for the definitive test were prepared by stirring an excess (150 mg/l) of test material in dechlorinated tap water at approximately 2000 rpm at a temperature of 25 degC for a period of 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration (0.2 microm) through a pre-conditioned filter to give a saturated solution of the test material at a nominal concentration of 12 mg/l from which dilutions were made to produce the remaining test groups of 0.67, 1.2, 2.1, 3.8 and 6.7 mg/l. Due to the flat response observed during the range-finding test, six test concentrations as opposed to five were employed during the definitive test to ensure a No Observed Effect Concentration (NOEC) would be obtained at 96 hours exposure.

- 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 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 13.0 to 14.4 degC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition period 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 espoused to the test material.
A semi-static test regime was employed in test involving a daily renewal of the test preparations to ensure that the concentrations of the test material 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.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% Confidence limit of 4.5 - 7.1 mg/l.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 2.1 mg/l, the lowest test concentration resulting in 100% mortality to be 12 mg/l and the No Observed Effect Concentration (NOEC) to be 2.1 mg/l.
Sub-lethal effects of exposure were observed at test concentrations of 3.8 mg/l and above. These responses were swimming at the bottom of the test vessels and loss of equilibrium.
After approximately 22 hours exposure three out of ten fish at 12 mg/l were observed to be moribund. At 6.7 mg/l two out of ten fish, one out of six fish and one out of four fish were observed to be moribund after approximately 45, 53 and 69 hours exposure respectively.
Due to the approach of the substantial severity limit these moribund fish were killed and classed as mortalities.
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 LC50 value of 5.6 mg/l with 95% confidence limits of 4.5 - 7.1 mg/l. The No Observed Effect Concentration (NOEC) was 2.1 mg/l.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1991-06-16 till 1991-09-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: "Letale Wirkung beim Zebrabärbling -Brachydanio rerio-" (LC 0 , LC 50 , LC 100; 48-96 Stunden) Verfahrensvorschlag: Umweltbundesamt Berlin, Stand Mai 1984
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
At defined concentrations of a substance arise after the test period different percentages of dead fish. The LC, 0 and LC 100 values thereby determined directly from the review, while the LC 50 value is determined by calculation or graphically from the dose-response curve. The test duration is 96 hours.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
-Concentrations: 250, 354, 500, 707 and 1000 mg/L
Analytical monitoring:
yes
Details on sampling:
daily inspection of the concentrations: 250 mg / L, 354 mg / L, 500 mg / L, 707 mg / L, 1000 mg / L and a control.
Vehicle:
no
Details on test solutions:
- direct weighing
- Dilution water: Reconstituted water prepared according to the recommendations of ISO. This freshly prepared standard dilution water was used for the maintenance of the test animals under flow-through conditions and for the preparation of stock- and test solutions of the test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Species: Zebrabärbling ( Brachydanio rerio HAMILTON BUCHANAN )
- Source: West-Aquarium ( Bad Lauterberg )
- Date of Birth: 1991-04-26
- Delivery: 1991-07-19
- Body length: 2.5 - 3.5 cm
- Medical treatment: no
- Maintenance water: synthetic freshwater according to ISO.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
13.2 °h
Test temperature:
temperature range: 20.9 - 21.7 °C
pH:
7.4 - 7.9
Dissolved oxygen:
- Control: 8.4 - 8.6 mg/L
- Test concentrations: 8.3 - 9.0 mg/L
Details on test conditions:
- Test vessel 300 x 135 x 200 mm
- Test medium: 5 L; ventilated
- static
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
707 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
About DOC analysis were at the weights of product 250 mg / L to 1000 mg / L of the dissolved fraction and the possible reaction products and metabolism products calculated daily.
These DOC values were at the test concentrations of 250 mg / L between 1 mg / L (= 2.0 mg / L test) and 8 mg / L (= 16.3 mg / L test), at 354 mg / L between 2 mg / L (= 4.1 mg / L test) and 9 mg / L (= 18.4 mg / L test), at 500 mg / L between 1 mg / L (= 2.0 mg / L test) and 15 mg / L
(= 30.6 mg / L test), at 707 mg / L from 4 mg / L (= 8.2 mg / L test) and 17 mg / L (= 34.7 mg / L test), at 1000 mg / L from 12 mg / L (= 24.5 mg / L test) and 18 mg / L (= 36.7 mg / L test).
Validity criteria fulfilled:
not specified
Conclusions:
The acute toxicity of 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt to fish was tested according to "Letale Wirkung beim Zebrabärbling -Brachydanio rerio-" (LC 0 , LC 50 , LC 100; 48-96 Stunden) Verfahrensvorschlag: Umweltbundesamt Berlin, Stand Mai 1984. A LC0 of 707 mg/L and a LC100 of >1000 mg/L were observed during 96 hours exposure (Bayer AG, 1982).
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990-04-02 til 1990-04-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions.
Qualifier:
according to guideline
Guideline:
other: "Letale Wirkung beim Zebrabärbling -Brachydanio rerio-" (LC 0 , LC 50 , LC 100; 48-96 Stunden) Verfahrensvorschlag: Umweltbundesamt Berlin, Stand Mai 1984.
Deviations:
yes
Remarks:
At the commencement of shares remained unresolved material at the surface of the test medium. The analytical concentrations found were some cases significantly below the target values ​​(<80 %), it was recorded in each case only the dissolved fraction.
Principles of method if other than guideline:
At defined concentrations of a substance arise after the testing period each percentages of dead fish. The LC will be 0 and 100 values ​​from this test determined directly, while the LC50-value calculation or graphically is determined from the dose-response curve. The test takes on 96 hours.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- direct weighing
- test concentrations: 7.8, 11, 16, 22, 31, 44 and 63 mg/L
- The analytical monitoring was performed in duplicate.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Type: zebrafish (Danio rerio HAMILTON BUCHANAN)
- Origin: West Aquarium (Bad Lauterberg)
- Date of birth: 1989-12-17
- Ship Date: 1990-03-07
- Body length: 2.5 - 3.5 cm
- Medical treatment: no
- Maintenance water: synthetic freshwater according to ISO
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Test temperature:
temperature range: 21.5 - 22.1 °C
pH:
7.4 -8.1
Dissolved oxygen:
7.8 - 9.0 mgO2/L
Nominal and measured concentrations:
- Test concentration: 7.8, 11, 16, 22, 31, 44 and 63 mg/L and control
- The analysis found concentrations were significantly lower than the nominal values. It was only the dissolved portions of the test substance can be detected
Details on test conditions:
- Test aquaria: 300 x 135 x 200 mm
- Test medium: 5 litre
- ventilated
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
other: LC0/LC100
Effect conc.:
37.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: geometric mean
Details on results:
At all concentrations remained unresolved material components in powder form on the surface of the test medium.
After 24 - 48 hours: Substance dissolved in part, unresolved material components are deposited on the walls of test vessels.
The above LC0 - or LC100-values refer to nominal concentrations, but above all were not reached, according to testing of chemical analytical monitoring.
Validity criteria fulfilled:
not specified
Conclusions:
Brachydanio rerio was exposed under static condition for 96 h to seven concentrations (7.8, 11, 16, 22, 31, 44 and 63 mg/L) of the test substance. The determination showed a LC0 at 22 mg/L and a LC100 at 63.0 mg/L, and the geometric mean LC0/LC100 was calculated at 37.2 mg/L.
Executive summary:

At defined concentrations of a substance arise after the testing period each percentages of dead fish. The LC0 and LC 100 values ​​from this test will be determined directly, while the LC50-value calculation or graphically is determined from the dose-response curve.

Brachydanio rerio was exposed under static condition for 96 h to seven concentrations (7.8, 11, 16, 22, 31, 44 and 63 mg/L) of the test substance. The determination showed a LC0 at 22 mg/L and a LC100 at 63.0 mg/L, and the geometric mean LC0/LC100 was calculated at 37.2 mg/L

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
37.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Validity criteria fulfilled:
not specified
Conclusions:
For the test item, the 96 h LC50 value for Brachydanio rerio was 37.2 mg/L.
Executive summary:

In a one-to-one read-across approach, the substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (source substance) is considered appropriate for direct read-across (one-to-one) to zinc 4-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide 7-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide (target substance) for the endpoint short-term toxicity to fish. In conclusion, the 96 h LC50 of the test item to Brachydanio rerio was 37.2 mg/L. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Description of key information

Key Study:

Short-term toxicity to fish (Oncorhyncus mykiss) 96 hour LC50 = 5.6 mg/L; OECD 203; Safepharm (2003).

Supporting Studies:

Short-term toxicity to fish (Bachydanio rerio) 96 hour LC0 = 707 mg/L and LC100 > 1000 mg/L; Letale Wirkung beim Zebrabärbling Verfahrensvorschlag: Umweltbundesamt 1984; Bayer AG 1992

Short-term toxicity to fish (Bachydanio rerio) 96 hour LC0 = 22 mg/L, LC50 37.2 mg/L and LC100 63 mg/L; Letale Wirkung beim Zebrabärbling Verfahrensvorschlag: Umweltbundesamt 1984; Bayer AG 1992 (data from read-across substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, used in a one-to-one approach)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
5.6 mg/L

Additional information

Key Study:

Safepharm, 2003- Examination of the test material structure,1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt (ZMB2), indicated that it was a zinc salt with the anion being a weak acid. It was therefore considered that the weak acid may associate with other cations of limited solubility leading to precipitation. As a result of this the maximum solubility of the test material in dechlorinated tap water was reduced by the presence of cations.

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, Safepharm, 2003, performed a modified version of the standard method for the preparation of aqueous media. An approach endorsed by several important regulatory authorities in the European Union and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (150 mg/L) of test material with dechlorinated tap water for 48 hours and then removing the undissolved test material by filtration through a pre-conditioned filter (0.2 µm) to give a saturated solution with a nominal concentration of 12 mg/L.

In accordance with OECD Guideline 203 (Fish, Acute Toxicity Test) the 96 -Hour LC50 based on nominal test concentrations of ZMB2 was 5.6 mg/L with 95% confidence limits of 4.5 - 7.1 mg/L. The No Observed Effect Level (NOEL) was 2.1 mg/L (Safepharm Laboratories, 2003).

Supporting studies:

Bayer 1992 - The acute toxicity of ZMB2 to fish was also tested according to "Letale Wirkung beim Zebrabärbling -Brachydanio rerio-" (LC 0 , LC 50 , LC 100; 48-96 Stunden) Verfahrensvorschlag: Umweltbundesamt Berlin, Stand Mai 1984. Fish were exposed to 250, 354, 500, 707 or 1000 mg/L of ZMB2. An LC0 of 707 mg/L and a LC100 of >1000 mg/L were observed during 96 hours exposure (Bayer AG, 1992).

The difference in acute toxicity between the Safepharm (2003) and Bayer (1992) study is expected to be due to the method used in the preparation of the test medium. In the Supporting study ZMB2 was incorporated directly into the test medium, with a LC100 of >1,000 mg/l. In the Key study it was highlighted that the maximum solubility of the test material in dechlorinated tap water was reduced by the presence of cations. In the Supporting study the concentrations used were 250, 354, 500, 707 and 1000 mg/L test material media preparation trials were conducted in order to determine the most suitable method of obtaining a saturated solution at a concentration similar to the water solubility level of 32 mg/L (Section 4.8 Safepharm, 2003).

Bayer 1990 - Brachydanio reriowere exposed to 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) at defined concentrations (7.8, 11, 16, 22, 31, 44 and 63 mg/L). The test was conducted in accordance with "Letale Wirkung beim Zebrabärbling -Brachydanio rerio-" (LC 0 , LC 50 , LC 100; 48-96 Stunden) Verfahrensvorschlag: Umweltbundesamt Berlin, Stand Mai 1984. No mortality occured at 7.8, 11, 16 and 22 mg/L, with 100 % mortality occuring at 63.0 mg/L. The LC50 was derived as the geometric mean of 22 and 63 mg/L (i.e. the LC0 and LC100 values). The LC50 was estimated to be 37.2 mg/L. However, analysis of the test item showed significntly lower exposure levels as only the dissolved portion stayed with the water column and precipatates were found at the surface fo the vessel. Therefore, the derived LC50 is not an accurate reflection of the toxicity of MB2 the endpoint can not be direclty compared to the ZMB2 result from the Safepharm (2003) study.

However, the study is consistent with the methodologies used in the Bayer (1992) study for ZMB2. It can be seen that MB2 is more toxic to fish in an acute fish toxicity test, the LC0 of MB2 = 22 mg/L and the LC100 was 63 mg/L and the LC0 for ZMB2 was 707 mg/L and the LC100 was > 1000 mg/L. Furthermore this also supports the fact that zinc is likely not the main driver of toxicity in an acute toxicity fish test.

Discussion:

As the Safepharm (2003) study uses the most appropriate methodology for a subtance of limited solubility, and the derived LC50 is the worst-case of the 3 tests, this value will be considered in the overall hazard assessment for the aquatic environment. It also highlights MB2 as a possible worst-case, although there were deficiency in the approach to testing for the MB2 test. This shows MB2 could be used as a worst-case in the overall assessment, abiding by the precautionairy principle.