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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:
2016/12/15-2017/12/13
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:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Batch number: 1366154001
- Purity: 98.7%
- Appearance / colour: solid/ yellow-brown
- Expiry date: 31 May 2019

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: 10.0, 5.00, 2.50, 1.25, 0.625 mg/L
- Sampling method: Analytical samples were taken in the main test at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from all loading rates and control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: All samples were stored deep frozen until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions (S1, S2, S3) were prepared by directly weighing 5.00 mg in 500 mL test medium, 5.00 mg in 1000 mL test medium and 2.50 mg in 1000 mL test medium. These stock solutions were stirred in the dark at room temperature for 24 h. The solutions were clear and transparent. Subsequently the necessary volumes for the test were withdrawn via Teflon tubes from the medium level of the stock solutions. The test item solutions V1 – V2 were made by diluting stock solution S3 with test medium without algae to give the required loading rates.
- Test concentrations: 10.0, 5.00, 2.50, 1.25, 0.625 mg/L and control

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 61.81
- Source: commercial supplier
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from commercial supplier.

ACCLIMATION
- Acclimation period: 3 to 4 days
- Culturing media and conditions: same as the test:
Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1);
Temperature: 21 - 24 °C;
Culture flasks: 100 mL Erlenmeyer flasks;
CO2 supply by shaking on a rotating shaker, approximately 105 rpm

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.3 – 22.8 °C
pH:
pH of control: 7.47 – 7.64
pH of test: 7.51 - 8.00
Nominal and measured concentrations:
Nominal concentrations: 10.0, 5.00, 2.50, 1.25, 0.625 mg/L
Measured concentrations (based on the geometric mean of each concentration level): 1.00, 0.424, 0.148, 0.0241 and 0.0638 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Renewal rate of test solution: no renewal
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 2.879E+05 /mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-medium

OTHER TEST CONDITIONS
- Photoperiod: Continuously from the side
- Light intensity and quality: 79.7 µEm-2s-1

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: fluorescence microplate reader, determined in duplicate at 24, 48 and 72 hours
- Other: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72 hour. The light intensity of all positions of the incubator is measured once a year and was confirmed for one representative position at test start. Analytical samples of all test item loading rates and control were analysed at test start (0 hours fresh) and at test end (72 hours aged).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: Control, 10.0, 1.00, 0.100, 0.0100 and 0.00100 mg/L
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test no concentration response relation was observed for the inhibition of growth rate and yield. The inhibition of growth rate peaked in 6.5 % at a nominal loading rate of 0.0100 mg/L and the inhibition of yield peaked in 20.5 % at a nominal loading rate of 0.0100 mg/L. (Results available in Any other information on results)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Biological Results: After 72 h at termination of the test no inhibition of growth rate or yield was observed for any of the employed loading rates up to and including 10.0 mg/L. The cells were considered normal for the control and up to and including a test item loading rate of 10.0 mg/L.
- Analytical Results: The measured initial concentration of test item ranged from 12 % to 18 % of nominal. In the aged samples the measured content was between < LOQ and 6 % of nominal.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: most recent test: yield 0.976 mg/L (nominal), growth rate 1.64 mg/L (nominal)
- Other: Significant inhibitory effects were determined for all parameters at test item concentrations of 0.800 mg/L and above.

Any other information on results incl. tables

Results non-GLP range finding test:

loading rate (mg/L) % inhibition at test end (72h)
growth rate yield
Control 0 0
0.001 2.8 10.7
0.010 6.5 20.5
0.1 3.4 11.2
1.0 1.4 6.4
10.0 -3.6 -12.6

Average cell number for each sampling time and loading rate:

Loading rate

Average cellnumbers[104/mL]

[mg/L]

0 h

24 h

48 h

72 h

Control

0.71

1.86

7.19

28.79

0.625

0.71

1.84

7.94

33.26

1.25

0.71

2.01

10.68

32.84

2.50

0.71

2.36

11.03

48.67

5.00

0.71

2.41

11.02

49.10

10.0

0.71

2.59

7.94

33.86

Percentage inhibition of growth rate:

Loading

rate

% Inhibition of growth rate

[mg/L]

0 h–24h

0h–48h

0h–72h

Control

0.0

0.0

0.0

0.625

1.9

-4.0

-3.7

1.25

-5.8

-17.2

-2.5

2.50

-25.2

-18.7

-14.3

5.00

-27.2

-18.6

-14.5

10.0

-34.5

-4.3

-4.5

Toxicological endpoints for the test item:

 

  Test item [mg/L]
Loading rate Measured Concentration
ErL10, ErC10(Growth rate) > 10.0 > 1.00
ErL20, ErC20 > 10.0 > 1.00
ErL50, ErC50 > 10.0 > 1.00
EyL10, EyC10(Yield) > 10.0 > 1.00
EyL20, EyC20 > 10.0 > 1.00
EyL50, EyC50 > 10.0 > 1.00
NOELR, NOEC 10 1
LOELR, LOEC - -

Determined concentration of Test item:

Test item nominal concentration (loading rate) [mg/L] Sampling Test item measured concentration [mg/L] % of nominal Geometric mean [%] Geometric mean concentration [mg/L]
Control 0 h fresh n.d. - - -
72 h aged n.d. -
0.625 0 h fresh 0.11 18 10.2 0.0638
72 h aged 0.0371 6
1.25 0 h fresh 0.232 19 1.93 0.0241
72 h aged < LOQ 2)
2.5 0 h fresh 0.441 18 5.93 0.148
72 h aged 0.0491 2
5 0 h fresh 0.611 12 8.48 0.424
72 h aged 0.293 6
10 0 h fresh 1.75 18 10 1
72 h aged 0.571 6

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
At test end, no statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 10.0 mg/L (nominal), corresponding to 1.00 mg/L (measured concentration). Thus the overall LOELR and LOEC were not determinable, the overall NOELR was observed to be at 10.0 mg/L (nominal) and the corresponding overall NOEC was determined as 1.00 mg/L (measured).
The EL10-, EL20- and EL50-value for growth rate (ErL10, 20, 50) and the EL10-, EL20- and EL50-value for yield (EyL10, 20, 50) were > 10.0 mg/L (nominal). The EC10-, EC20- and EC50-value for growth rate (ErC10, 20, 50) and the EC10-, EC20- and EC50-value for yield (EyC10, 20, 50) were > 1.00 mg/L (measured).
Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD Guideline 201.

A static test with nominal loading rates of 10.0, 5.00, 2.50, 1.25, 0.625 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration.

Samples from control and all test item loading rates at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions were analysed. The measured initial concentration of test item ranged from 12% to 18% of nominal. In the aged samples the measured content was between < LOQ and 6% of nominal. The corresponding geometric mean concentrations were 0, 0.0638, 0.0241, 0.148, 0.424 and 1 mg/L.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 10.0 mg/L (nominal) and 1.00 mg/L (measured concentration) at test end. Thus the overall LOELR and LOEC were not determinable, the overall NOELR was observed to be at 10.0 mg/L (nominal) and the corresponding overall NOEC was determined as 1.00 mg/L (measured).

The EL10-, EL20- and EL50-value for growth rate (ErL10, 20, 50) and the EL10-, EL20- and EL50-value for yield (EyL10, 20, 50) were > 10.0 mg/L (nominal). The EC10-, EC20- and EC50-value for growth rate (ErC10, 20, 50) and the EC10-, EC20- and EC50-value for yield (EyC10, 20, 50) were > 1.00 mg/L (measured).