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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 21, 1997 to April 24, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
HPLC method
Details on sampling:
Sampling schedule:
At the start and at the end of exposure
Vehicle:
yes
Remarks:
Hardened castor oil (HCO-40)
Details on test solutions:
For both pre-culture and testing, the medium specified in the OECD Chemicals Test Guidelines was prepared and used after sterilization.

The test substance was dissolved in the medium to make 2.0 wt%, which was sterilized by filtration to obtain the test substance stock solution. Each test solution was prepared by adding the test substance stock solution to a filter-sterilized medium. Four test vessels were used for each concentration group and control group. One of them was used for pH measurement at the start of the test. A medium was used as a control group. The test solution was colorless and transparent, and no precipitation was observed.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Exposure method : Static, shaking intensity (100 rpm)
- Specimen : Selenastrum capricornutum (ATCC 22662)
- Test water temperature : 23 ± 2 °C
- Test solution amount : 100mL (OECD medium)
- Lighting : 4.512~4.532 lux (Continuous lighting)
- Initial cell concentration : 1 x 10 E04 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
23.0 °C overall the entire period
pH:
The pH of the test solution was 7.2 ~ 7.4 at the start of exposure and 7.8 ~ 9.5 at the end of the test
Dissolved oxygen:
No data
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
Test concentration (n): 0.02 mg/L, 0.05 mg/L, 0.13 mg/L, 0.32 mg/L, 0.80 mg/L, 2.0 mg/L
Details on test conditions:
1 Test conditions
The test was conducted under the following conditions. However, a sterilized test container was used, and algae was inoculated under sterile conditions.

1) Culture method: Shake culture (100 rpm)
2) Temperature: 23 ± 2 °C
3) Exposure period: 72 hours
4) Test solution volume: 100 mL (OECD medium)
5) Lighting: 4.512~4.532 lux (Continuous lighting)
6) pH: No pH adjustment during the exposure period.
7) Initial cell concentration: 1 x 1004 cells / mL

2 Medium
For both pre-culture and testing, the medium specified in the OECD Chemicals Test Guidelines was prepared and used after sterilization.
3 Test container, algae culture test equipment and Equipment
- Test container: 300 mL glass Erlenmeyer flask (with air permeable silicone stopper)
- Algae culture test equipment: Ito Seisakusho AGP-150RL
- Optical microscope: Nikon culture inverted microscope TMS-F
- pH meter: HORIBA, Ltd. Castany LAB pH meter F-22
- Particle counter: Coulter Coulter Z1
- Electrolyte for particle counter: Isoton n
- Thermometer: Alcohol thermometer
- Luminometer: Tokyo Optical Machinery Digital Illuminometer IM-2D

4 Setting of test concentration
Prior to the implementation of this test, a preliminary test was conducted in which three concentration groups (common ratio 5) of 0.08 mg/L, 0.4 mg/L, and 2.0 mg/L were set, and EbC50 was obtained by the area method (0-72h). It was estimated to be 0.31 mg/L. Based on the results of this preliminary test, in this test, the common ratio was 2.5, and the solvent control group, 0.02 mg/L, 0.05 mg/L, 0.13 mg/L, 0.32 mg/L, 0.80 mg/L.
2.0 mg/L was set in 6 levels..



Reference substance (positive control):
yes
Remarks:
Potassium dichromate, special grade reagent
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Since the toxicity value for 0 to 72 hours by the algae growth rate was not reported in the reports before 2003, it is calculated from the original data.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Since the toxicity value for 0 to 72 hours by the algae growth rate was not reported in the reports before 2003, it is calculated from the original data.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Area under growth curve
Remarks on result:
other: 95%- confidence limit: 0.396~0.656 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.016 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Area under the growth curve
Results with reference substance (positive control):
The 72-hour growth inhibition concentration (EbC50) of the reference substance (potassium dichromate, special grade reagent) was 0.48 mg/L.
Reported statistics and error estimates:
I. Calculation of no-effect concentration (NOEC)
The highest test concentration at which no significant difference (5% level) was observed compared to the control group by the statistical method (homoscedasticity test and test of difference in mean value) was defined as the no-effective concentration (NOEC).


II. Statistical methods used
- Probit-Method: The Probit-Method program (Version 3.1) developed by Yoshimasa Yoshioka, a professor at Gifu Medical Junior College (currently a professor at the Faculty of Education, Oita University) to calculate IC50 and EC50 in the Ecological Impact Test of the Environment Agency, is used as it is. EC50 was calculated.
- Homoscedasticity test (F test): Prior to the test for the difference in mean values, the test for homoscedasticity is performed according to the following procedure, and the test method for the difference in mean values is selected based on the test results.
Validity criteria fulfilled:
yes
Conclusions:
The growth inhibition of N-Cyclohexyl-2-benzothiazolylsulphenamide (95-33-0) to algae was investigated in a static GLP study following OECD 201. After 72 hours of exposure the EC50 value was 0.500 mg/L and the NOEC 0.016 mg/L by comparison of the area under the growth curve. Since the toxicity value for 0 to 72 hours by the algae growth rate was not reported in the reports before 2003, it is calculated from the original data (https://www.nite.go.jp/chem/jcheck/template.action?ano=1644&mno=5-0256&cno=95-33-0&request_locale=ja). The resulting EC50 based on growth rate was 0.15 mg/L and the NOEC 0.0084 mg/L. The above concentrations are all based on analytical values.
Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 201 “Algae growth inhibition test”. A growth inhibition test was conducted on algae (Selenastrum capricornutum) to determine the growth inhibition concentration (EC50) and no-effect concentration (NOEC) under static exposure conditions. The number of cells of the pre-cultured algae was counted, and a certain amount of the pre-culture solution was added to the container containing the test solution so that the cell concentration in the test solution was 1×104 cells/mL. Each test container is placed in a culture device at 23 ± 2 °C to start the test, and the cell concentration is measured 24, 48 and 72 hours later. After collecting mL and mixing it with an electrolytic solution (Isotonne 1) to make a total volume of 20 mL, the measurement was carried out with a coulter counter. The pH at the time of preparing the test solution was measured only for one spare bottle prepared in addition to the triplet, and was used as the pH at the start of exposure in each concentration group. At the end, all three stations were measured, and the average value was taken as the pH at the end of the test. During the test period, the temperature and illuminance in the incubator were measured once a day. A preliminary test was conducted prior to the implementation of this test, and EbC50 (0-72h) was estimated to be 0.31 mg/L. Based on the results of the preliminary test, 6 levels of concentration groups of 0.02 mg/L, 0.05 mg/L, 0.13 mg/L, 0.32 mg/L, 0.80 mg/L, 2.0 mg/L were set in this test. After 72 hours of exposure the EC50 value was 0.500 mg/L and the NOEC 556 mg/L by comparison of the area under the growth curve. Since the toxicity value for 0 to 72 hours by the algae growth rate was not reported in the reports before 2003, it is calculated from the original data (https://www.nite.go.jp/chem/jcheck/template.action?ano=1644&mno=5-0256&cno=95-33-0&request_locale=ja). The resulting EC50 based on growth rate was 0.15 mg/L and the NOEC 0.0084 mg/L. The above concentrations are all based on analytical values. The 72-hour growth inhibition concentration (EbC50) of the reference substance (potassium dichromate, special grade reagent) was 0.48 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the algae growth inhibition study.

Description of key information

The toxicity of CBS to aquatic algae (Selenastrum capricornutum) was tested according to OECD TG 201 "Alga, Growth Inhibition Test". After 72 hours of exposure an ECr50 of 0.15mg/L and a NOEC of 0.0084 mg/L were obtained (MOE Japan, 1997).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.15 mg/L
EC10 or NOEC for freshwater algae:
0.008 mg/L

Additional information

A hydrolysis half-life time of CBS of 12.5 h and a complete hydrolysis of CBS in 24.9 h were observed in deionized water buffered at pH 7.0 and at 25°C (Monsanto, 1984). The effect concentrations in the study by the National Institute of Technology and Evaluation Japan (MOE, 1997) is based on the measured concentration at the start of the exposure. Hardened castor oil (HCO-40) was used as a vehicle.


The absolute values of chlorophyll and/or cell numbers were not reported for control experiment in Monsanto study from 1979 and hence no exponential growth can be concluded from this study. Therefore the information provided in this study is not sufficient for assessment. Although this study was used in EU-risk assessment (2008), this study is assigned to be a reliability of 4 in the up-to-date assessment and will not be used in the risk assessment under REACH.


CBS can be rapidly degraded by hydrolysis and the main degradation products appearing during the time frame of acute tests are benzothiazole (BT) and benzothiazolone (BTon). The toxicities of BT and BTon to alga were tested according to OECD TG 201(AKZO NOBEL, 2004). The 72h-ErC50 (nominal) was 50.8 mg/l (BT) and 22.4 mg/l (BTon); and the NOEC was 19.4 mg/l (BT) and 9.9 mg/l (BTon), respectively. The nominal concentration was confirmed analytically; however the results should be used provisionally with reservations due to lack of exponential growth in the control cultures. There are some other benzothiazole derivatives as metabolites from CBS are considered as relevant for environmental exposure and risk assessment, such as MeBT, MBT, MBTS and MeSBT. The toxicities of MeBT and MeSBT to alga were tested according to OECD TG 201 and the 72h- ErC50 of 45.2 mg/l and 3.43 mg/l (nominal) were determined, respectively, which were analytically confirmed (AKZO NOBEL, 2004). Since the control cultures did not grow exponentially, in EU-risk assessment (2008), the data were re-evaluated. According to re-evaluation of the raw data and expert judgement, an ErC50 = ca. 32 mg/l is considered more defendable for MeBT. For MeSBT the re-evaluated value is ca. two times higher than the reported value, and hence the reported value was considered sufficiently protective and provisionally used with reservations. The toxicity of MBT to aquatic algae (Selenastrum capricornutum) was tested according to OECD TG 201 "Alga, Growth Inhibition Test". After 72 hours of exposure, an ECr50 of 0.5mg/L and a NOEC of 0.066 mg/L were obtained (MITI, 1999). For MBTS no acute toxic effect was observed to algae (Desmodesmus subspicatus) up to its water solubility (Bayer AG, 1992). Regarding the data summarized here, the described metabolites of CBS are less toxic to aquatic algae than CBS itself.