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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to GLP and following OECD guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
A volume of test sample was diluted (1:1) with methanol and analyzed.

Standard solutions of test material were prepared in methanol/water* (50:50 v/v) at a nominal concentration of 10mg/l.

*Prepared by ELGA Ultima water purification.
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.

In each of five ground glass stoppered conical flasks an amount of test material (75 mg) was dispersed in reconstituted water (500 mL) to give initial test material dispersions at a concentration of 150 mg/L. The test material dispersions were shaken (INFORS Aerotron) at 300 rpm at a temperature of 30 degC for a period of 48 hours. After the shaking period the contents of the replicate flasks were pooled, cooled to 21 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. Aliquots (5, 9, 16, 28, 50, 90, 160 and 280 ml) of this saturated solution were each separately dispersed in a final volume of 500ml of reconstituted water to give the remainder of the test series.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

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. 4.8 Safepharm 2002 gave water solubility value of 32 mg/L. It was therefore considered that filtration was an appropriate method for the removal of undissolved test material.

- Method 1:
An amount of test material (1650 mg) was dispensed in 11 liters of reverse osmosis purified deionized water with the aid of propeller stirring at approximately 2000 rpm at a temperature of 21 degC for 48 hours to give an initial test material dispersion of 150 mg/L. After 48 hours the stirring 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 23 mg/L.

- Method 2:
An amount of test material (75 mg) was dispersed in 500ml of reverse osmosis purified deionised water with the aid of shaking (INFORS Aerotron) at approximately 300 rpm at a temperature of 30 degC for 48 hours. After 48 hours the shaking was stopped and the undissolved test material was removed by filtration (0.2 micm 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):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 liters of reconstituted water in a temperature controlled room at 12 degC. The lighting cycle was controlled to give a 16 hours light and 8 hours dark cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnias produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
21 degC.
pH:
pH of 7.8 +/- 0.2
Nominal and measured concentrations:
The mean measured test concentrations were: 0.13, 0.25, 0.47, 0.86, 1.6, 2.6, 5.0, 8.8 and 15 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. This was considered possibly to be due to the filters used in the media preparation trials not being 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.

In each of four round glass stoppered conical flasks amounts of test material (75 mg) were dispersed in reconstituted water (500 ml) to give initial test material dispersion at a concentration of 150 mg/L. The test material dispersions were shaken at 300 rpm at a temperature of 30 degC for a period of 48 hours. After the shaking period the contents of the replicate flasks were pooled and any undissolved test material was removed by filtration (0.2 microm Gelman Suporcap filter, first approximate 1 liter discarded to pre-condition the filter) tp produce 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 concentration in the saturated solutions was 8.0 mg/L. This value was lower than the test concentration of 21 mg/L determined by DOC analysis during the media preparation trial when reverse osmosis purified deionized 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. It was therefore considered that the weak acid may associate with the other cations of limited solubility leading to precipitation. As a result of this the maximum solubility of the test material in reconstituted water was reduced by the presence of cations. These cations were not present in the reverse osmosis purified deionized water used in media preparation trials or the distilled water used in 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 8.0 mg/L. Serial dilutions were made from this saturated solution to give test concentrations of 0.80, 0.080, and 0.0080 mg/L. However care should be taken in the interpretation of the concentration of the saturated solution and thus the concentrations for the range-finding test as the saturated solution prepared for the definitive test was determined to be 15.7 mg/L.

In the range-finding test 10 daphnias were placed in each test and control vessel and maintained in a temperature controlled room at 21 degC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minutes dawn and dusk transition periods. Each 250 mlLtest and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test material.

Definitive test:
Based on the results of the range-finding test and the preliminary recovery analyses conducted, the test material solutions for the definitive test were prepared by shaking an excess (150 mg/L) of test material in reconstituted water at approximately 300 rpm at a temperature of 30 degC for a period of 48 hours. After 48 hours the shaking was stopped and any undissolved test material was removed by filtration (0.2 micro) through a pre-conditioned filter to give a saturated solution of the test material at a nominal concentration of 8.0 mg/L from which dilutions were made to produce the remaining test groups of 0.080, 0.14, 0.25, 0.45, 0.80, 1.4, 2.5 and 4.5 mg/lL However care should be taken in the interpretation of the concentration of the saturated solution and thus the concentrations for the range-finding as the saturated solution prepared for the definitive test was determined to be 15.7 mg/L.

Exposure conditions:
As in the range-finding tests 250mL glass jars containing approximately 200ml of test preparation were used. At the start of the study 10 daphnia were placed in each test and control vessel at random in the test preparations. Duplicate test vessels were used for each test and control groups. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 degC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minutes dawn and dusk transition periods. The daphnia were not individually identified received no food during exposure and the test vessels were not aerated.

The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to the exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits of 1.1 - 1.6 mg/l.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.47 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range finding test:
No immobilization was observed at the nominal test concentrations of 0.0080 and 0.080 mg/L. However immobilization was observed at 0.80 and 8.0 mg/L.

Based on this information the test material solutions for the definitive test were prepared by shaking an excess (150 mg/L) of test material in reconstituted water for a period of time and then removing any undissolved test material by filtration to give a saturated solution. This saturated solution was then further diluted, as necessary, to produce the remainder of the test series.

Definitive test:
The immobilization observed at 24 hours indicated a flat response of the test organisms to the test material with 10%, 45%, 75%, 65% and 60% immobilization being observed at mean measured concentrations of 1.6, 2.6, 5.0, 8.8 and 15 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of ZMB2 to the freshwater invertebrate Daphnia magna has been investigated and based on the mean measured test concentrations gave a 48 hour EC50 value of 1.4 mg/L with 95% confidence limits of 1.1 - 1.6 mg/L. The No Observed Effect Concentration at 48 hours was 0.47 mg/L.
Endpoint:
short-term toxicity to aquatic invertebrates
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 document.
Reason / purpose:
read-across source
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits: 1.56 - 2.34 mg/L
Validity criteria fulfilled:
not specified
Conclusions:
For the test item, the 48 h EC50 value for Daphnia magna was 1.9 mg/L (95 % confidence limits: 1.56 - 2.34 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 invertebrates. In conclusion, the 48 h EC50 of the test item to Daphnia magna was 1.9 mg/L (95 % confidence limits: 1.56 - 2.34 mg/L). A full justification for the read-across approach is presented in IUCLID Section 13.2.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011-06-22 till 2011-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Principles of method if other than guideline:
The method described in the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004) assesses the acute toxic effects (immobilisation) of various concentrations of a test item to a freshwater microcrustacean species.
The purpose of this method was to determine that concentration which causes a 50 % immobilisation rate (= EC50) or, if conducted as a limit test, to determine the acute toxic effects at a maximum test concentration of 100 mg/L or at the limit of water solubility.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Concentration of 100 mg/L was measured at 0 and 48 h; and concentration of 0 mg/L at 48 h only
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
A stock solution was prepared to give the desired series of test concentrations. 10.4 mg of the test item were added to 1 litre of dilution water and treated for 1 hour in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Finally undissolved particles of the test item were removed by an aseptic filter (0.2 µm). The pH was measurd to be 8.3.
The concentration of the test item was determined analytically by HPLC to be 9.796 mg/L.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 19 mL per replicate. 1 mL of dilution water containing 10 daphnids was given to all replicates resulting in the final nominal concentrations. For each test item concentration and the control 2 replicates were prepared.
Test organisms (species):
other: Daphnia magna STRAUS, parthenogenetic females, Strain of Bundesgesundheitsamt Berlin
Details on test organisms:
Maintenance and Acclimatisation: A population of parthenogenetic females of synchronized age structure has been maintained for more than 15 years in the test facility under constant temperature conditions (20 +/- 1 °C) at a 16 : 8 hour light-dark photoperiod (light intensity: < 20 µE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily in a semi- quantitative way. The neonates were separated from their parent Daphnia by filtration prior to the acute test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
14.9°dH (= 266 mg/L CaCO3)
Test temperature:
19.9 - 20.5 °C (+/- 1 °C) measured at each test vessel at the beginning and the end of the test
pH:
8.0 - 8.1 measured at each test vessel at the beginning and the end of the test
Dissolved oxygen:
7.7 - 9.2 mg O2/L with 84 - 101 % saturation measured at each test vessel at the beginning and the end of the test
Nominal and measured concentrations:
Test item concentration: 0.08, 0.17, 0.38, 0.83, 1.8 and 4 mg/L plus control
The results are expressed in terms of nominal concentrations at 24 and 48h. Effective concentrations ranged from 88.75 % to 99.40 % of nominal values at 0 hours and from 80.0 % to 100.75 % of nominal values at 48 hours.
Details on test conditions:
PRETREATMENT OF TEST ITEM.
- 10.4 mg of the test item addedto 1 litre od dilution water
- treated for 1 hour in an ultrasonic bath
- stirred for 24 h on a magnetic stirrer
- 19 mL of the solution were taken and diluted with 1 mL of dilution water resulting in a final concentration of 9.796 mg/L

TEST SYSTEM:
- Test vessels: 50 mL glass beakers covered with watch glasses holding 10 neonates in 20 mL of test medium
- Experimental design: 6 test concentrations plus 1 control
10 neonates per vessel, 2 replicates per concentration/control
no feeding during the exposure period static system
- Method of initiation: neonates were placed in prepared media
- Photoperiod: 16 h light : 8 h dark
- Temperature of incubation unit: 20.1 °C
- Aeration: none
- Test item concentration/s: 0.08, 0.17, 0.38, 0.83, 1.8 and 4 mg/L
- Method of administration: stock solution
- Medium renewal: none
- Duration of exposure: 48 hours
- Criteria of effects: The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits: 1.56 – 2.34 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Validity criteria fulfilled:
yes
Remarks:
(The immobilisation and other abnormalities in the controls did not exceed 10 % by the end of the test. The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period.)
Conclusions:
Daphnia Magna was exposed under static condition for 48 h to six concentrations (0.08, 0.17, 0.38, 0.83, 1.8 and 4 mg/L) of the test substance. The effect concentration was determined as EC50 = 1.90 mg/L.
Executive summary:

In order to test acute toxicity to invertebrates of the substance, Daphnia Magna, was exposed to the test solution of six concentrations of the test substance ( 0.08, 0.17, 0.38, 0.83, 1.8 and 4 mg/L) and blank control solution for a period of 48 h under static conditions.Mobility and visible abnormalities were recorded at 24 and 48 h. The measured concentrations confirmed that deviation from the nominal concentrations was less than 21 % (measured concentrations were in the range of 80.0 % to 100.75 % of nomianl concentrations). An effect concentration showed an EC50 = 1.90 mg/L. The toxcitity study is classified as accetable and satisfies the guideline requirements for the acute Daphnia study.

Description of key information

Key Study:

Short-term toxicity to aquatic invertebrates (Daphnia magna) 48 hour EC50 = 1.4 mg/L; OECD 202; Safepharm (2003).

Supporting study:

Short-term toxicity to aquatic invertebrates (Daphnia magna) 48 hour EC50 = 1.9 mg/L; OECD 202; Currenta (2001) (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

EC50/LC50 for freshwater invertebrates:
1.4 mg/L

Additional information

Key study:

Safepharm, 2003 - In accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) the acute toxicity of ZMB2 to the freshwater invertebrate Daphnia magna was investigated.

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 test medium 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 23 mg/L.

The Daphnia magna were exposed to concentrations of 0.13, 0.25, 0.47, 0.86, 1.6, 2.6, 5.0, 8.8 and 15 mg/L. Daphnia magna were exposed under static conditions for 48 hours. Mobility was recorded after 48 hours. The 48 hour EC50 was estimated to be 1.4 mg/L (mean measured) with 95% confidence limits of 1.1 - 1.6 mg/L. The 48 hour No Observed Effect Concentration (NOEC) is 0.47 mg/L.

Supporting study:

Currenta, 2011 - In order to test acute toxicity to invertebrates of the substance, Daphnia Magna, were exposed to a test solution of six concentrations of the MB2 (0.08, 0.17, 0.38, 0.83, 1.8 and 4 mg/L) and blank control solution, for a period of 48 h under static conditions. Stock solutions were prepared by adding 10.4 mg to 1 litre of test medium and then being treated in an ultrasonic bath for 1 hour, the mixture was then stirred for 24 hours. Undissloved particles were removed using a 0.2 µm filter. Mobility and visible abnormalities were recorded at 24 and 48 h. The measured concentrations confirmed that deviation from the nominal concentrations was equal to or less than 20 % (measured concentrations were in the range of 80.0 % to 100.75 % of nomianl concentrations). Therefore, the EC50 was based on nominal concnetrations. An effect concentration showed an EC50 = 1.90 mg/L. The toxcitity study is classified as accetable and satisfies the guideline requirements for the acute Daphnia study.

Discussion:

It can be seen that MB2 and ZMB2 have a similar toxicological effect on Daphnia magna during short-term toxicity testing. This supports the use of MB2 as a read-across source substance for ZMB2.

As data are available on the target substance this will be considered during the overall hazard assessment of ZMB2.