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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 24 Aug. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, Annex 5 corrected: 2011

Deviations:

yes

Remarks:

Details are described within the section "Any other information on material and method incl tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test item concentrations
- Sampling method: The content of phosphorus (total P and P from orthophosphate) of the test item in the test solutions were measured at the beginning and at the end of the test.
- Sample storage conditions before analysis: Samples were taken every 24 h and stored at -20 ± 5 °C.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing 176.0 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. This was done by filling 111 µL test item with algal medium to 1000 mL. This solution was shaken by hand for homogenization. The solution was clear. Lower test concentrations were prepared by dilution of the stock solution in algal medium.
- Controls: Yes, medium only

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green alga
- Strain: SAG 86.81
- Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen
- Age of inoculum (at test initiation): 5 days
- Method of cultivation: Kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 5 days
- Culturing media and conditions (same as test or not): Conditions for the pre-culture was the same as under testing conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.7 – 23.2 °C

pH:

8.1 - 8.7 (Control)
8.1 - 9.2 (Test item concentrations)

Nominal and measured concentrations:

Control, 8.1, 18, 39, 81, 176 mg/L (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: Glass flasks total volume 65 mL, filled with 45 ± 1 mL test solution
- Initial cells density: 2.2 *10E^03 cells/mL
- Control end cells density: 537.413 *10E^03 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD 201 medium
- Culture medium different from test medium: Yes, stock culture was growing on solid agar at 2 - 8 °C, pre-culture was growing in the same medium as the test culture

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continously
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter; every 24 h, the cell number was determined with an electronic particle counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study: Non-GLP pre-test was performed

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 92.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: calculation via constituent 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 20.15 - < 22.93 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: calculation via constituent 3 and 1

Remarks:

for details see tables in section "Any other information on results incl table"

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): The lowest concentration (8.1 mg/L nominal) is not used for evaluation, since an agglomeration of the algae cells was observed in some replicates.
- Any stimulation of growth found in any treatment: At 18 mg/L positive inhibition values were measured as a sign for stimulating effect at this test item concentration

Results with reference substance (positive control):

The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72 h EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.

Analytical Results:

The test item consists of 3 constituents. In this study, the content of the test item in the test solutions was determined once by the concentration of constituent 1 and once by the concentration of constituent 3.

Constituent 3: The content of P from orthophosphate in constituent 3 (potassium sodium ortho-phosphate) could be measured using UV-VIS (method 1). Constituent 3 has a P content of 15.6% (calculated using molecular weight). The test item consists of 16.8% constituent 3, so that constituent 3 can be used to calculate back to the test item concentration.

Constituent 1:

The total P content of constituents 1 and 3 could be measured using the ICP-OES (method 2). If the UV-VIS value (method 1) is subtracted from this total P content, the P content of constituent 1 is obtained. Constituent 1 has a P content of 8.5% (calculated by molecular weight). The test item consists of 22.4% constituent 1, which means that constituent 1 can be used to calculate back to the test item concentration.

At test start (0 h) a correction factor of QC-sample of 92.0% was taken into account.

At test end (72 h) a correction factor of QC-sample of 95.1% was taken into account.

In both cases, the concentrations determined deviate by slightly more than ±20% from the nominal value. Therefore the geometric mean is formed. Geometric mean is calculated by multiplication of the n participating concentrations and taking the nth root. The water content of 43.1% is deducted from these values and the concentration of active ingredient (a.i.) is calculated.

Biological Results:

Table 1: Results of the test item (based on active ingredient, calculation via constituent 1, best-case)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	9.20 mg/L	25.60 mg/L	22.93 mg/L	> 102.7 mg/L
Yield	9.20 mg/L	25.60 mg/L	16.87 mg/L	31.97 mg/L

 

Table 2: Results of the test item (based on active ingredient, calculation via constituent 3, worst-case)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	8.70 mg/L	18.00 mg/L	20.15 mg/L	> 92.9 mg/L
Yield	8.70 mg/L	18.00 mg/L	11.44 mg/L	28.10 mg/L

 

Table 3: Validity criteria:

Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	244	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	17 %	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2 %	Yes

Validity criteria fulfilled:

yes

Remarks:

See section "Any other information on results incl tables"

Description of key information

 

Results of the test item (based on active ingredient, calculation via constituent 3, worst-case)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	8.70 mg/L	18.00 mg/L	20.15 mg/L	> 92.9 mg/L
Yield	8.70 mg/L	18.00 mg/L	11.44 mg/L	28.10 mg/L

 

In absence of a calculated EC50 value the threshold is set to the maximum concentration of 92.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

92.9 mg/L

EC10 or NOEC for freshwater algae:

20.15 mg/L