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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2017 - 21 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals, Beijing: SEPA
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems. The 2nd edition. Beijing: China Environment Press. 2013: 6-21.
Version / remarks:
2nd edition, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21805-2008, Chemicals-Alga growth inhibition test, Beijing: SAC
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of 10 mL were taken from the blank control and the limit concentration for analysis at t=0, t=24, t=48 and t=72 h.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test substance was added directly to AAP medium in glass aspirator bottle to give the required loading rates. The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 24 hrs on a magnetic stirplate with a Telfon stirbar at room temperature. At the end of the 24 h, the mixtures were left to settle for 1 h at room temperature. Then the aqueous portion was removed through the outlet of the aspirator stirring bottle.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source: Freshwater Algae Culture Collection at the Institute of Hydrobiology (Chinese acadamy of science), Lot No: A20171015P.

ACCLIMATION
- Acclimation period: a pre-culture was started 4/5 days before the start of the test
- Culturing media and conditions: same as in the test. The pre-culture was incubated on a shaker with 3000 Lux (±15%) continuous illumination at 23.5°C, and the rotor speed was 100 (±10) rpm.
- Any deformed or abnormal cells observed: cultures were checked every day during pre-culture, no deformed cells, fungus or other algae were observed.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
23.1°C - 23.6°C throughout the test
pH:
7.15-7.61 (controls) and 7.09-7.51 (test solutions) throughout the test
Nominal and measured concentrations:
Nominal concentration: WAF prepared at a loading rate of 100 mg/L
Measured TOC concentrations: 24.0 mg/L at t=0 h, 20.6 mg/L at t=24 h, 22.2 mg/L at t=48 h, 21.2 mg/L at t=72 h
Since measured concentrations were considered to remain stable throughout the test period, the mean measured test concentration was calculated to be 22.0 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass flasks with air-permeable stoppers, fill volume: 100 mL
- Initial cells density: 2.10 × 10^4 cells/mL
- Control end cells density: 423 × 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium by mixing appropriate volumes of different stock solutions and diluting these with deionized water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, temperature was also measured throughout the test.

OTHER TEST CONDITIONS
- Sterile test conditions: for the preparation of inoculum culture sterile medium was used.
- Adjustment of pH: the pH of the AAP medium was adjusted to 7.5 ± 0.1 with 0.1 N HCl.
- Photoperiod: no
- Light intensity and quality: continuous, uniform fluorescent illumination of cool-white type at 4989~5193 Lux

EFFECT PARAMETERS MEASURED: growth rate at 24 h, 48 h and 72 h
- Determination of cell concentrations: by microscope at 24 h, 48 h and 72 h
- Other: microscopic observations at the end of the test to observe any abnormal appearance of the algae

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: based on the results of the range-finding study only a limit test at 100 mg/L was performed.
- Range finding study
- Test concentrations: WAFs prepared at 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, after 72 hours of exposure, the growth of the algae cultures exposed to the highest concentration was inhibited for 2.08% compared to the blank control. The EC50 was therefore expected to exceed 100 mg/L in the final limit test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- After 72 hours of exposure, growth was inhibited for 0.61% in limit concentration of a WAF prepared at 100 mg/L.
- The measured concentrations of the test substance in medium were between 20.6 and 24 mg/L and were considered stable during the test period. Therefore, effect concentrations were based on the mean measured concentration (22 mg/L).
- No abnormalities were observed during microscopic observations at the end of the test period.
- Biomass in the blank control and the treatment groups increased exponentially.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h-ErC50: 3.00 mg/L (95% CI: 2.86-3.16)
Reported statistics and error estimates:
The EC50 and 95% confidence limits were calculated by Trimmed Spearman-Karber Method (Version 1.5, USEPA). For estimation of the NOEC the Independent Samples T-test (SPSS 19.0) was used.

Any other information on results incl. tables

Table 1 Growth inhibition during the limit test

Nominal

Con.

0h

24h

48h

72h

pH

*CellCon.

Inhibition Rate(%)

*CellCon.

Inhibition Rate(%)

pH

*CellCon.

Inhibition Rate(%)

0

blank control

7.16

23.2

147

7.52

426

-

7.22

24.1

150

7.61

420

7.20

24.1

139

7.50

412

7.23

23.2

149

7.53

440

7.15

24.1

142

7.55

422

7.18

23.2

140

7.52

419

specific growth rate(day-1)

2.420 ~ 1 d

2.120 ~ 2 d

1.770 ~ 3 d

Coefficientsof variation

0.840 ~ 1 d

0.800 ~ 2 d

0.420 ~ 3 d

specific growth rate(day-1)

2.420 ~ 1 d

1.811 ~ 2 d

1.072 ~ 3 d

Coefficientsof variation

0.840 ~ 1 d

2.391 ~ 2 d

2.322 ~ 3 d

100 mg/L WAFs

7.11

18.8

13.0

120

4.86

7.41

413

0.61

7.12

15.3

112

7.45

407

7.09

17.1

124

7.37

423

7.15

18.0

119

7.49

402

7.14

15.3

120

7.51

420

7.13

19.7

111

7.49

392

ErC50(mg/L)

(based on measured concentration)

> 100mg/L WAFs

> 100mg/L WAFs

> 100mg/L WAFs

*(×104cells/mL)

Table 2 Measured TOC Concentrations

Nominal Concentration

(mg/L)

Measured TOC Concentration(mg/L)

Mean

(mg/L)

0h

24h

48h

72h

blank control

3.48

3.38

3.53

3.64

3.51

100 mg/L WAFS

16.0

14.1

15.1

14.7

15.0

Deduct blank control TOC value

12.5

10.7

11.6

11.1

11.5

Concentration of test substance

24.0

20.6

22.2

21.2

22.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks' section.
Conclusions:
In the toxicity to algae study, performed according to OECD guideline 201 and GLP principles, the NOEC of STABiO D-376N was determined to be 22 mg/L, the 72h-ErC50 was >22 mg/L.
Executive summary:

A study was performed according to OECD guideline 201 and GLP principles to determine the toxicity of STABiO D-376N towards algae. Six replicate cultures of Pseudokirchneriella subcapitata were exposed to the test substance for 72 hour. Test solutions were prepared as Water Accomodated Fractions (WAFs) for a dose range-finding study and a limit study. In the dose-rang finding study, WAFs prepared at loading rates of 1.0, 10 and 100 mg/L were used. No significant inhibition of growth was observed at the highest tested concentration and therefore a limit study was performed to determine the effect concentrations. Samples of the test solution were taken at t=0, t=24, t=48 and t=72 h to determine actual exposure concentrations. Analysis showed that the concentration of test substance in the test solution remained stable over the exposure period. Therefore, the mean measured concentration was calculated to be 22 mg/L. To determine the inhibition of growth microscopic observations were performed. No significant inhibition of growth was observed at the limit concentration. The NOEC and 72h-ErC50 of STABiO D-376 were determined to be 22 mg/L and >22 mg/L based on the mean measured concentration.

All validity criteria were met and the study was considered to be valid.