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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:

Experimental phase: 18 January 2018 to 02 March 2018: Report issued: 21 August 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)

Deviations:

yes

Remarks:

A minor deviation from the study plan was not considered to have any impact on the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Culture: The master cultures were maintained in the laboratory under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: None, test cultures were seeded direct from the master cultures.
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type : closed (The flasks were plugged with polyurethane foam bungs)
- Fill volume: 100mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 5.00 x 10^3 cells per mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux, warm white light 380 to 730 nm.


TEST CONCENTRATIONS
- Range finding study: Two range finding tests conducted
- Test concentrations: Test 1: Nominal loading rates of 10 and 100 mg/L. Test 2: Nominal loading rates of 0.01, 0.1 and 1 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Run as a separate study using potassium dichromate as the positive control.

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Results with reference substance (positive control):

A separate positive control study is run periodically in the laboratory using potassium dichromate to confirm that the algae respond as expected. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L LOading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loadong Rate (LOEL) (mg/L
Growth Rate	0.87	-	0.32	1.0
Yield	0.43	0.38 - 0.49	0.10	0.32

Validity criteria fulfilled:

yes

Conclusions:

Based on the water accommodated fraction (WAF) the EL50 (mg/L loading rate WAF) was 0.87 mg/L based on growth rate and 0.43 mg/L based on yield. The No Observed Effect Level (NOEL) was 0.32 mg/L based on growth rate and 0.1 mg/L based on yield. The Lowest Observed Effect Level (LOEL) was 1.0 mg/L based on growth rate and 0.32 based on yield.

Executive summary:

Introduction

The study was performed to assess the effect of the test item on the growth of the green alga. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

Method

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following preliminary range-finding tests, the green alga Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.016 mg/L, to 0.70 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the LOQ in all test preparations.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L LOading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loadong Rate (LOEL) (mg/L
Growth Rate	0.87	-	0.32	1.0
Yield	0.43	0.38 - 0.49	0.10	0.32

Conclusion

Based on the water accommodated fraction (WAF) the EL50 (mg/L loading rate WAF) was 0.87 mg/L based on growth rate and 0.43 mg/L based on yield.  The No Observed Effect Level (NOEL) was 0.32 mg/L based on growth rate and 0.1 mg/L based on yield.  The Lowest Observed Effect Level (LOEL) was 1.0 mg/L based on growth rate and 0.32 based on yield.

Description of key information

Test substance toxicity to fresh water algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.87 mg/L

EC10 or NOEC for freshwater algae:

0.32 mg/L