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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April 2020 - 01 May 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Test solutions prepared using OECD GD 23 (Water Accommodated Fraction)
Principles of method if other than guideline:
Test concentrations used were WAFs individually prepared at a loading of 0.10 to 1.0 µg/L
Minor deviations from the guideline: 1. The solvent control had 5 replicate vessels instead of 6. 2. At the end of the test, no algae were observed under the microscope. These deviations were not considered to impact the integrity of the study or interpretation of the study results and conclusions.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: stock solutions from the ecotoxicity test were diluted with dichloromethane if necessary, to obtain concentrations within the calibration range.
- Sample storage conditions before analysis:Stored at room temperature
Vehicle:
yes
Remarks:
Dichloromethane, however this was evaporated completely overnight before addition of test medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution of 1.0 µg/mL was further diluted to obtain the stock solutions used to prepare the test solutions.
- Eluate: Not reported
- Differential loading: Yes - empty test vessels were individually spiked with 1.0mL of the respective stock. Each replicate of the solvent control received 1.0mL DCM. The solvent was completely evaporated overnight before addition of the test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent. Then, 50 mL of the test medium was added to each vessel.
- Controls: both test media control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dichloromethane
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions including those of the blank and the solvent control, were agitated for a period of two days to ensure maximum dissolution of the test item in test medium.
- Other relevant information:
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in house laboratory culture
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to a light in a climate room at a temperature of 21-24 degrees C


- Culturing media and conditions (same as test or not): different to test media. Stock culture medium was M1 (according to Nederlandse Praktijk Richtlijn no. 6505). Test medium was M2 (according to OECD 201 guideline).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
22-24 degrees C
pH:
7.6 - 8.0
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 0.10 and 1.0 µg/L
Measured: 0.087 and 0.78 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100mL all glass vessels with aluminium caps, perforated for ventilation
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50mL test solution with 50mL headspace
- Aeration: Not aerated, but during incubation, the algal cells were kept in suspention by continuous shaking
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 1 x 10(4) cells/mL
- Control end cells density: 355 x10(4) cells/mL. Solvent control: 367 x10(4) cells/mL
- No. of organisms per vessel: initial cell density of 1 x 10(4) cells/mL
- No. of vessels per concentration (replicates): 6 replicates at highest test concentration, 3 replicates of the intermediate test concentration
- No. of vessels per control (replicates): 6 replicates for blank control,
- No. of vessels per vehicle control (replicates):5 replicates for solvent control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium (made according to OECD 201)
- Total organic carbon: Not reported
- Particulate matter:Not reported
- Metals:Not reported
- Pesticides:Not reported
- Chlorine:Not reported
- Alkalinity:Not reported
- Ca/mg ratio: Hardness as Ca+Mg 0.24 mmol/L (24 mg CaCO3/L)
- Conductivity:Not reported
- Culture medium different from test medium:Yes, test medium is M2, culture medium is M1
- Intervals of water quality measurement: 0h and 72h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:Media adjusted to 8.1 +/- 0.2
- Photoperiod: continuous lighting
- Light intensity and quality: 60 to 120 μE/m2/s when measured in wavelength range of 400 - 700 nm
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber for initial cell densities. Cell densities thereafter were determined by spectrophotometric measurement using a spectrophotometer with immersion probe
- Chlorophyll measurement: Not measured
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor of 10
- Justification for using less concentrations than requested by guideline: was a limit test/range finding test so only used two concentrations
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, test conducted 11 May - 18 June 2020, non-GLP and algae showed expected sensitivity to reference substance.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 0.78 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 0.78 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 0.78 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: No stimulation of growth was observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: None reported
Results with reference substance (positive control):
The batch of Raphidocelis subcapitata tested, showed expected sensitivity to Potassium dichromate in a study conducted between 11 May 2020 and 18 June 2020. No further details were given.
Reported statistics and error estimates:
A student t-test (parametric, α=0.05, two-sided) was performed to test for presence of statistically significant differences in algal growth between the blank and solvent control. The ELx-values could not be determined since the observed effects were below 10%.

Table 1. Mean cell densities of P. subcapitata during exposure of Di-tert-Nonyl Polysulfide 537

Time (hours)

Cell densities (x104cells/mL)

(Mean (SD))

Blank control

Solvent control

0.10

1.0

0

1 (0)

1 (0)

1 (0)

1 (0)

24

13.292 (0.434)

13.271 (0.369)

N.d.

13.209 (0.269)

48

75.960 (3.624)

76.720 (3.774)

N.d.

74.457 (4.083)

72

354.987 (30.507)

367.409 (16.861)

347.507 (29.711)

350.324 (18.462)

Table 2. Mean growth rates of P. subcapitata during exposure of Di-tert-Nonyl Polysulfide 537

Time (hours)

Growth rates (day-1)

(Mean (SD))

Blank control

Solvent control

0.10

1.0

0-24

2.587 (0.033)

2.585 (0.028)

N.d

2.581 (0.020)

0-48

2.165 (0.024)

2.170 (0.024)

N.d

2.154 (0.027)

0-72

1.956 (0.030)

1.969 (0.015)

1.949 (0.029)

1.953 (0.018)

Validity criteria fulfilled:
yes
Remarks:
All three validity criteria outlined in OECD TG 201 were met.
Conclusions:
72-hour EL50, EL10 and NOELR values of >0.78, >0.78 and ≥0.78 µg/L, respectively have been reported for the effects of the test substance on the growth rate of the freshwater alga, Pseudokirchneriella subcapitata (reported as Raphidocelis subcapitata), based on measured concentrations. The study met the OECD TG 201 validity criteria and was conducted according to GLP, however the preparation of the stock solutions was not sufficiently described. Therefore the study has been assigned a reliability score of 2.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
14/06/99 - 06/03/00
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, Guideline Study - one exception to guideline due to nature of test material
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
As saturation concentration less than detection limit it was not possible to meet the validity criterion regarding the stability of the test substance.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
not specified
Details on sampling:
Algal concentrations were measured daily. At test initiation and termination, analytical and physico-chemical measurements were also carried out.
Vehicle:
yes
Details on test solutions:
Vehicle, solvent: dilution water completed with NaHCO3 (0.3 g/L) and Hepes buffer state (6mM). Since TPS is poorly soluble in water; a saturated solution was prepared by vigorously mixing 100 mg of polysulfide di-tert-dodecyl with 1 litre of dilution water during 24 hours (this solution was agitated at 30°C for 3 hours and then at 23°C for 21 hours). After this saturation period, droplets were observed on glass and in suspension in water. Solution appearance was visually checked at test initiation and termination, with solutions being clear and colourless, with no precipitation observed. The saturated solution was filtered with a 0.22 µm filter.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain: CCAP 278/4. Source/supplier: Culture Centre of Algae and Protozoa. Method of cultivation: Parent cell suspensions were subcultured every week in 100 mL flasks, incubated at 23 ± 1°C, with alternative lighting (16h light / 8h darkness), under continuous aeration with filtrated air (0.22µm). Pretreatment: 4 days before treatment, parent cultures are diluted (5 mL in 500 mL) to prepare 2 pre-cultures (one for sowing, the other for rescue). Controls: Quality of parent cultures was checked under a microscope to verify the absence of microorganisms or deformed cells. Initial cell concentration: 10000 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
No data reported
Test temperature:
24 ± 1°C
pH:
At test start: 7.16 - 7.28
At test end: 7.61 - 7.68
Dissolved oxygen:
At test start: 9.0 - 9.3 mg/L
At test end: 10.0 - 10.5 mg/L
Salinity:
No data reported
Nominal and measured concentrations:
Saturation, 0.080 mg/L. Concentrations: 9.39, 20.66, 45.45, 100 nominal concentration (% vol) forming a geometric progression with a factor of 2.2.
Details on test conditions:
Test type: phytoculture chamber with rotating trays. Renewal of test solution: No. Exposure vessel type: 120 mL glass bottles stoppered with PTFE bungs and sealed with aluminum caps. Number of replicates per dose: 3. Photoperiod: 16h light / 8h darkness. pH and dissolved oxygen were measured at the beginning and the end of the test in the test solutions.
Reference substance (positive control):
no
Remarks:
Current method (C3, 92/69/EEC) does not require a test with K2Cr2O7. However, the sensibility of the biologic reactive is checked by a toxicity test with K2Cr2O7 every 2 months.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: Limit of solubility
Details on results:
Algae appeared normal at test termination in comparison with the control when checked by microscopic observation. No precipitation was observed at test termination. The results of this study show that no inhibition of growth was observed after 72 hours of exposure to the saturated stock solution of polysulfide di-tert-dodecyl.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Statistical test: Dunnett test.
Validity criteria fulfilled:
no
Remarks:
The growth in the control was greater than that required for validity. As saturation concentration less than detection limit it was not possible to meet the validity criterion regarding the stability of the test substance.
Conclusions:
The toxicity of polysulfide di-tert-nonyl to algae has been read across from polysulfide di-tert-dodecyl. 72 hours of exposure of polysulfide di-tert-dodecyl at its saturation concentration (0.08 mg/L) was found not to inhibit the growth of Pseudokirchnerella subcapitata .
Executive summary:

The toxicity of polysulfide di-tert-nonyl to algae has been read across from polysulfide di-tert-dodecyl. Thiebaud (2000) is a GLP-compliant guideline study showing the growth inhibition effect of polysulfide di-tert-dodecyl on Pseudokirchnerella subcapitata over 72 hours. The maximum level of exposure, the saturation concentration (0.080 mg/L), resulted in no inhibition of the growth of Pseudokirchnerella subcapitata. The study is considered reliable and suitable for use for this endpoint.

Description of key information

72-hour EL50, EL10 and NOELR values of >0.78, >0.78 and ≥0.78 µg/L were reported for the effects of the registration substance on Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

Additional information

72-hour EL50, EL10 and NOELR values of >0.78, >0.78 and ≥0.78 µg/L, respectively have been reported for the effects of the registration substance (CAS 68425-16-1, EC 270-336-2) on the growth rate of the freshwater alga, Pseudokirchneriella subcapitata (reported as Raphidocelis subcapitata), based on measured concentrations. The study was conducted according to OECD TG 201, met the validity criteria and was conducted according to GLP. Due to the low water solubility of the test substance, the test solutions were prepared as Water Accommodated Fractions (WAFs).

Additionally, a supporting study has been included for the analogue substance polysulfide di-tert-dodecyl. Thiebaud (2000) is a GLP-compliant guideline study showing the growth inhibition effect of polysulfide di-tert-dodecyl on Pseudokirchnerella subcapitata over 72 hours. The maximum level of exposure, the saturation concentration (0.080 mg/L), resulted in no inhibition of the growth of Pseudokirchnerella subcapitata. The study is considered reliable.