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Ecotoxicological information

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:
22 Apr 2016 to 27 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
March 2006
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): zinc bis(dibenzyldithiocarbamate)
- Batch No.: 50705100
- Date of receipt: 19 October 2015
- Expiration date: 31 July 2017
- Appearance: withe to light cream powder
Analytical monitoring:
yes
Details on sampling:
After 24 and 48h of incubation, aliquot of 200 μL was sampled from each inoculated test flask, pipetted into a microplate. After 72 h, the samples with high cell density were dilute to ¼ (50 μL + 150 μL of filtered dilution water) and pipetted into a microplate. Microplates were then analysed using a flow cytometer (Guava easyCyte™ flow cytometer Merck Millipore). Time between sampling and measurement is approximately 15 - 30 min.
Vehicle:
no
Details on test solutions:
the stock solution was prepared at 1 mg/L loading rate and stirred during around 24 hours in order to achieve the saturation concentration, then diluted to get the required test solutions. Non-dissolved particles were removed from the test medium per filtration through a 0.45 μm membrane filter.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source: The Culture Centre of Algae and Protozoa (Ambleside, UK).
- Method of cultivation: Four days before the start of the exposure, two pre-cultures were prepared by inoculating each stock suspension of algae into sterile dilution water. The pre-cultures were incubated under the same conditions as those used for the test. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only used if the first one was damaged.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
19.7 - 20.9 °C
pH:
7.9 - 8.2
Dissolved oxygen:
7.9 - 9.2 mg O2/L
Nominal and measured concentrations:
- Saturated solution concentrations: 0 (control), 10, 17.2, 30.8, 55.6 and 100% v/v saturated test susbtance solution
An overview of the measured concentrations is presented in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were made of glass bottles (120 mL capacity). They were filled with a volume of 50 mL. The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions; flasks were continuously shaken with a rotation at around 100 rpm.
- Type: closed, capped with cellulose bungs.
- Fill volume: 50 mL
- Initial cells density: ca 0.5 x 10E+04 cells/mL.
- Control end cells density: 33.64E+04 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- Abiotic control: Yes, one satellite non-inoculated vessel per test concentration (containing only dilution water and test item at the requested concentration, including a control) was incubated under the same test conditions.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous
- Light intensity and quality: Fluorescent light tubes, 6,000 and 10,000 lux.

WATER QUALITY MEASUREMENTS
The pH and dissolved oxygen concentrations were measured in all inoculated test solutions at the beginning and the end of the test. The temperature in the incubator was continuously recorded throughout the test.

EFFECT PARAMETERS MEASURED: cell density
Algal cell concentrations were measured in each flask at 24, 48 and 72 h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. The cytometer is calibrated every week using the Guava check kit. Before each measurement, settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than alga were used to assess the toxic effects. The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell concentration was less than 500 cells/μL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore).
Reference substance (positive control):
yes
Remarks:
The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No effects observed. The ErC50 was determined as greater than the maximum solubility of test item.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No effects observed. The ErC10 was determined as greater than the maximum solubility of test item.
Details on results:
An overview of the results is presented in 'Any other information on results incl. tables'.
- Appearance of the test substance: The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.
- Algae appearance: Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 μm.
- Control growth curve: The examination of plots shows exponential growth of control culture at the expected rate throughout the test.
- Growth rate: No statistically significant inhibitory effect on the growth rate of the algae after the test period of 72 hours at all the tested concentrations.
- Yield: No statistically significant inhibitory effect on the yield of the algae after the test period of 72 hours at all the tested concentrations.
Results with reference substance (positive control):
The latest value of ErC50 (obtained in March 2016) was determined to be 0.97 mg/L.
Reported statistics and error estimates:
The growth inhibition data were analysed using an Excel program. NOEC, LOEC, EC10 and EC50 values as well as the 95% confidence intervals were then determined using the software ToxRat® according to the recommendations of the guideline.

Table: Average percentages of inhibition of growth rate and yield

Nominal concentration

(% v/v saturated solution)

Growth rate (%)

Yield (%)

10.0

-0.8

-3.3

17.2

1.5

6.2

30.8

-0.4

-1.5

55.6

0.3

1.2

100.0

-3.9

-18.3

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.

Description of key information

72-h ErC50: >100% (v/v) (Pseudokirchneriella subcapitata)

72-h ErC10: >100% (v/v) (Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (Arkema, 2018). In this study, exponentially growing freshwater alga (Pseudokirchneriella subcapitata) were exposed to saturated solutions of the test substance of 0, (control),10, 17.2, 30.8, 55.6 and 100% (v/v) for 72 hours under static conditions. The test was performed in three replicates per test concentration. The initial concentrations and the maintenance of the exposure concentrations during the test were analytically determined. Cell density was measured after 24, 48 and 72 hours exposure and the growth rate, yield and biomass per time point were determined. Throughout the test, no effects on the growth rate and yield were observed.Based on these findings, the 72-h EC50 values for growth rate and yield/biomass are determined to be >100% v/v. The 72-h ErC10 was also determined to be >100% v/v for both endpoints.