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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-Aug-2019 to 26-Sep-2019
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:
2006, Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all groups.
Frequency: at t=0 h and t=72 h.
Volume: 2.0 mL from the approximate centre of the test vessels.
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WSF prepared at a loading rate of 3.2 mg/L but without algae and samples for analysis were taken at the start and at the end of exposure.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. To this end, the according test item amounts were weighed onto watch glasses and added to flasks containing 1 L test medium. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning and used as test concentrations. Lower test concentrations were prepared by subsequent dilutions of the WSF prepared at a loading rate of 1.0 mg/L in test medium.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata.
- Strain: NIVA CHL 1.
- Source: In-house laboratory culture.
- Age of inoculum: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- 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 light in a climate room at a temperature of 21-24°C.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
between 22 and 23°C
pH:
8.0 - 8.3
Dissolved oxygen:
not applicable
Salinity:
not applicable
Conductivity:
not applacable
Nominal and measured concentrations:
Nominal concentrations: WSFs individually prepared at loading rates of 1.0, 3.2, 10 and 32 mg/L, and solutions containing 10 and 32% of the WSF prepared at a loading rate of 1 mg/L.
Measured concentrations:
- The measured concentrations of the mono-ester were 0.092, 0.32, 0.98, 3.3, 9.9 and 32 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L.
The concentrations remained generally stable, i.e. the 1.0 to 32 mg/L WSFs were at 82-94% relative to initial at the end of the test, however the 10 and 32% dilutions of the 1.0 mg/L WSF decreased to 72-73% of initial at the end of the test.
- The measured concentrations of the di-ester were 0.010, 0.039, 0.13, 0.40, 1.2 and 3.4 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L.
The concentrations remained stable, i.e. were at 100-111% relative to initial at the end of the test.

Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
- Aeration: No
- Initial cells density: An initial cell density of 1 x 10^4 cells/mL.
- Control end cells density: A mean cell density in the controle of 253 x 104 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: yes.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 80 to 85 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED :
- Effect parameters: 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50, 72 h ErC20, 72 h EyC20, 72 h ErC10, 72 h EyC10.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Test medium was used as blank.

TEST CONCENTRATIONS
Combined Limit/Range-Finding Test:
- Test Concentrations: Exponentially growing algal cultures were exposed to WSFs prepared at loading rates of 1.0, 10 and 100 mg/L and to a control.
- Results were used to determine the conditions for the definitive study: Yes.


Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
initial measured concentration of di-ester (m/z 321.1)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
initial measured concentration of di-ester (m/z 321.1)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 0.18 to 0.30 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
initial measured concentration of di-ester (m/z 321.1)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 1.1 to 1.4 mg/L.
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test reve aled a normal and healthy appearance of the algal cells exposed to the limit concentration when com pared to the control.
Results with reference substance (positive control):
Results with reference substance valid?: yes
The 72h-EC50 for growth rate inhibition (ERC50) was 2.02 mg/L with a 95% confidence interval ranging from 2.00 to 2.04 mg/L.
Reported statistics and error estimates:
For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding initially measured concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Initial Measured concentrations

The substance

WSF of loading rate (mg/l)

  10% of WSF 1.0 mg/L  32% of WSF 1.0 mg/L  WSF of 1.0 mg/L  WSF of 3.2 mg/L  WSF of 10 mg/L  WSF of 32 mg/L

 Measured concentration

mono-ester (m/z209.1) (mg/L)

0.092   0.32 0.98  3.3  9.9  32 
 Measured concentration di-ester (m/z321.1) (mg/L) 0.010  0.039  0.13  0.40  1.2  3.4 

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period

The substance
Initial Measured conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.844

0.0185

6

 

0.010

1.833

0.0107

3

0.62

0.039

1.839

0.0189

3

0.29

0.13

1.798

0.0355

3

2.5

0.40

1.153

0.1328

3

 37*

1.2

1.402

0.0813

3

 24*

3.4

0.086

0.1493

3

 95*

* Effect was statistically significant.

 

Table 3: Growth Rate and Percentage Inhibition at Different Time Intervals

 

The substance
Initial Measured conc. (mg/L) 

n

0 – 24h

24 – 48h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.192

 

1.707

 

1.634

 

0.010

3

2.140

2.3

1.733

-1.5

1.626

0.53

0.039

3

2.157

1.6

1.660

2.8

1.701

-4.1

0.13

3

2.038

7.0

1.716

-0.49

1.641

-0.44

0.40

3

1.153

47

0.791

54

1.516

7.2

1.2

3

1.469

33

1.218

29

1.520

7.0

3.4

3

0.359

84

-0.359

121

0.259

84

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, the substance inhibited growth rate of this freshwater algae species significantly at initial measured concentrations of 0.40 mg/L di-esther and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.4 mg/L.
The 72h-NOEC for growth rate inhibition was 0.13 mg/L.
The ERC10 for growth rate inhibition was 0.24 mg/L.



Executive summary:

A 72 hour toxicity study with freshwater algae (Pseudokirchneriella subcapitata ) was concducted according to OECD guideline 201 and GLP principles. The algae were exposed to WSFs individually prepared at loading rates of 1.0, 3.2, 10 and 32 mg/L, and solutions containing 10 and 32% of the WSF prepared at a loading rate of 1.0 mg/L.

Samples taken from all test concentrations and the control were analysed by following the mono-ester (m/z209.1) and di-ester (m/z321.1). The measured concentrations of the mono-ester were 0.092, 0.32, 0.98, 3.3, 9.9 and 32 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L. The concentrations remained generally stable, i.e. the 1.0 to 32 mg/L WSFs were at 82-94% relative to initial at the end of the test, however the 10 and 32% dilutions of the 1.0 mg/L WSF decreased to 72-73% of initial at the end of the test.

The measured concentrations of the di-ester were 0.010, 0.039, 0.13, 0.40, 1.2 and 3.4 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L. The concentrations remained stable, i.e. were at 100-111% relative to initial at the end of the test.

Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario.

The substance inhibited growth rate of this freshwater algae species significantly at measured concentrations of 0.40 mg/L and higher.The 72h-EC50for growth rate inhibition (ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.4 mg/L. The 72h-NOEC for growth rate inhibition was 0.13 mg/L.The 72h-EC10 for growth rate inhibition was 0.24 mg/L.

The study met the acceptability criteria, and is considered to be valid.

Description of key information

Fresh water algae study, 72 Hours, ERC10 = 0.24 mg/L, ERC50 = 1.2 mg/L, NOEC = 0.13 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
1.2 mg/L
EC10 or NOEC for freshwater algae:
0.13 mg/L

Additional information

A 72 hour toxicity study with freshwater algae (Pseudokirchneriella subcapitata ) was conducted according to OECD guideline 201 and GLP principles.

The algae were exposed toWSFs individually prepared at loading rates of 1.0, 3.2, 10 and 32 mg/L,and solutions containing 10 and 32% of the WSF prepared at a loading rate of 1.0 mg/L.

Samples taken from all test concentrations and the control were analysed by following the mono-ester (m/z 209.1) and di-ester (m/z 321.1).The measured concentrations of the mono-ester were 0.092, 0.32, 0.98, 3.3, 9.9 and 32 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L. The concentrations remained generally stable, i.e. the 1.0 to 32 mg/L WSFs were at 82-94% relative to initial at the end of the test, however the 10 and 32% dilutions of the 1.0 mg/L WSF decreased to 72-73% of initial at the end of the test.

The measured concentrations of the di-ester were 0.010, 0.039, 0.13, 0.40, 1.2 and 3.4 mg/L at the start of the test, respectively to the 10 and 32% dilutions of the 1.0 mg/L WSF and the loading rates of 1.0, 3.2, 10 and 32 mg/L. The concentrations remained stable, i.e. were at 100-111% relative to initial at the end of the test.

Based on these results, effect parameters were expressed as initially measured concentrations of the di-ester, since the lower measured concentrations represent the worst case scenario.

The substance inhibited growth rate of this freshwater algae species significantly at measured concentrations of 0.40 mg/L and higher.The 72h-EC50 for growth rate inhibition (ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.4 mg/L. The 72h-NOEC for growth rate inhibition was 0.13 mg/L. The 72h-EC10 for growth rate inhibition was 0.24 mg/L.

The study met the acceptability criteria, and is considered to be valid.