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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:
The range-finding test was conducted between 9 and 12 June 2015 and the definitive test between 20 and 23 July 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study was conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006; Annex 5 corrected 28 July 2011)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD series on testing and assessment number 23, 2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples (15 mL) were taken from the control and each exposure level at the start of the test and after 24 and 72 hours.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Samples were stored in the freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: saturated solution
Jasmal was not completely soluble in test medium at the loading rate initially prepared, as a layer of undissolved material was observed at the surface of the solution.
* test medium: M2
* loading rate: 100 mg/L
* stirring period: 70-88 minutes of magnetic stirring in a vessel to reach the maximum solubility of the test substance in the test medium
* phase separation: after stirring, the aqueous phase was separated from the undissolved layer observed at the surface of the test solution by siphoning and used as the highest test concentration (range-finding test)
* test concentration range: lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium

TEST CONCENTRATIONS:
* control (no test substance) and solutions containing 10, 18, 32, 56 and 100% of the saturated solution prepared at a loading rate of 100 mg/L
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

ACCLIMATION
- Stock culture medium: M1
- Pre-culture:
* Combined limit/range-finding test: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
* Final test: because the pre-culture did not grow sufficiently, test solutions were inoculated with algae originating from the stock culture

After preparation, volumes of 50 mL test solution were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
Temperature was maintained between 22 and 24°C throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.9 - 8.2.
Nominal and measured concentrations:
Range-finding test - nominal test concentrations: 0.1, 1.0 and 10% of a saturated solution prepared at 100 mg/L.

Final test:
Based on the results of the range-finding test the following nominal test concentrations were assigned to the final test: 10, 18, 32, 56 and 100% of a saturated solution prepared at 100 mg/L.
Measured test concentrations: see table 1 in field "any other information on results'

The effect concentrations are based on time-weighted average measured concentrations.


Details on test conditions:
TEST SYSTEM
- Test vessel:
100 mL all-glass vessels were used, each containing 50 mL of test preparation for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.

- No. of vessels per concentration (replicates): 3 + 1 extra replicate of each test concentration for sampling purposes at t=24
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 1 (at 32% of the saturated solution)

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: M2 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore), pH adjusted to 8.1 ± 0.2)

- llumination: continuously using TLD-lamps with a light intensity within the range of 75 to 76 µE.m-2.s-1 while constantly shaking.

- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities were determined. 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 720 nm using a spectrophotometer with cuvettes (path length =10 mm. Algal medium was used as blank.

- Appearance of the cells: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to
observe for any abnormal appearance of the algae.

- Results used to determine the conditions for the definitive study:
The results of the range-finding test showed that growth was reduced by 31% in tests solutions at 100% of the saturated solution prepared at 100 mg/L. Based on this information test concentrations of 10, 18, 32, 56 and 100% of a saturated solution prepared at 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (June 2015)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
58 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 49-72 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 6.7-11 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities in algal cells: no

Statistically significant inhibition of growth rate was found at TWA concentrations of 7.0 mg/L and higher, however, the effect observed at 7.0 mg/L was considered biologically insignificant (<10%). Therefore this value is used as NOEC. In none of the test concentrations inhibition of growth rate >= 50% was reached.

Statistically significant inhibition of yield was observed in all test solutions, ranging from 20% in the lowest to 87% inhibition in the highest treatment group.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.3 mg/L (95% confidence interval ranging from 1.2 to 1.5 mg/L)
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOEC and the EC50, 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 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 average exposure concentrations of the test substance.

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Measured test substance concentrations

Samples taken from all concentrations were analysed. Despite the fact that the test substance seemed to be not completely dissolved after magnetic stirring, the actual concentrations measured at the start of the test were at the level of nominal (89-101%). After 24 hours of exposure, the actual concentrations were at the level of 74-88% of nominal, with an exception of the solution incubated without algae and containing 32% of saturated solution. In this sample the actual concentration decreased to 51% of the nominal. At the end of the test the measured concentrations were at the level of 0.42-11% of nominal, with exception of the samples containing 32% of saturated solution, in which the measured concentration was below the limit of detection of the analytical method. Because this result was unexpected, the reserve samples were analysed. The measured concentrations were at the level of 0.41-9.3% of nominal but this time the actual concentration in the solution containing 56% of saturated solution was below the limit of detection. The reason for these observations is unknown. Therefore, for the calculation of TWA concentrations an average concentrations measured in the samples taken at the end of the test were used. Where applicable, the concentration was set as half of the LOD.

Table 1: Concentrations of the test item in test medium - final test

Time of sampling
[hours]

Percentage of SS2

[%]

Concentration

Analysed1
[mg/l]

Relative to initial
[%]

 

 

 

 

0

0

n.d.

 

 

10

10.1

 

 

18

16.1

 

 

32

31.2

 

 

 323

31.6

 

 

56

54.0

 

 

100

93.1

 

 

 

 

 

24

0

n.d.

n.a.

 

10

7.45

74

 

 104

8.19

81

 

18

15.4

95

 

32

28.2

90

 

 323

16.3

51

 

56

47.3

88

 

100

82.1

88

 

 

 

 

72

0

n.d.

n.a.

 

10

 0.0425

0.42

 

18

0.521

3.2

 

32

< LOD6

n.a.

 

 323

< LOD6

n.a.

 

56

1.38

2.6

 

100

10.6

11

 

 

 

 

724

0

n.d.

n.a.

 

10

 0.0415

0.41

 

18

0.411

2.6

 

32

 0.0475

0.15

 

 323

 0.0265

0.082

 

56

< LOD6

n.a.

 

100

9.29

10

 

 

 

 

1          Samples were stored in the freezer (≤ -15°C) until the day of analysis (31-Jul-2015).

2          Percentage of a water saturated solution (SS) prepared at a loading rate of 100 mg/L.

3          Without algae.

4          Spare samples, analysed on 11-Aug-2015

5          Estimated value, calculated by extrapolation of the calibration curve.

6          The limit of detection of the method was determined to be 0.038 mg/L taking a concentration factor of 5 into account.

n.d.    Not detected.

n.a.     Not applicable.

Table 2: Measured concentrations versus nominal concentrations

Jasmal

% saturated solution prepared at 100 mg/L

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72 h

First run

Second run

10

10.1

7.795#

0.042

0.041

3.3

18

16.1

15.4

0.521

0.411

7.0

32

31.2

28.2

0.019*

0.047

11

32 WA

31.6

16.3

0.019*

0.026

8.0

56

54

47.3

1.38

0.019*

21

 100

93.1

82.1

10.6

9.29

48

WA – without algae

* - half of the LOD (LOD=0.038 mg/l)

#- mean of two measurements

 

Because the actual concentrations in the solutions incubated without algae showed similar responses, it can be concluded that the decrease of the concentrations was not associated with the growing algal biomass.

Inhibition results

Table 3: Percentage inhibition of growth rate (total test period) during the final test

Jasmal

TWA concentration

(mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.446

0.0551

6

3.3

1.371

0.0701

3

5.2

7.0

1.356

0.0555

3

6.2*#

11

1.261

0.0269

3

12.8*

21

1.091

0.0458

3

24.5*

48

0.793

0.1031

3

45.2*

* - effect was statistically significant

#- effect biologically insignificant (<10%)

Table 4: Percentage inhibition of yield during the final test

Jasmal

TWA concentration

(mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

76.5

13.19

6

3.3

60.9

12.38

3

20.4*

7.0

58.0

9.32

3

24.2*

11

43.1

3.63

3

43.7*

21

25.6

3.57

3

66.6*

48

10.1

3.63

3

86.7*

* - effect was statistically significant

Validity criteria fulfilled:
yes
Remarks:
In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%
Conclusions:
The ErC50, ErC10 and NOEC were 58, 8.8 and 7.0 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed for 72 hours to 5 concentrations of the test substance, prepared from a saturated solution of the test material prepared at 100 mg/L (three replicate flasks per concentration), under constant illumination and shaking at a temperature of 22 - 24°C. The test material solutions were prepared by magnetic stirring at room temperature for 70 - 88 minutes followed by siphoning off the aqueous phase to exclude undissolved material.

Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. Measured concentrations at test initiation ranged from 89 - 101% of nominal, and decreased to 78 - 88% of nominal after 24 hours and 0.07 - 10% of nominal at test end (mean of 2 measurements). Therefore time-weighted average (TWA) concentrations were calculated and used for expression of endpoints. Nominal concentrations of 10, 18, 32, 56 and 100% of the saturated solution prepared at 100 mg/L corresponded with 3.3, 7.0, 11, 8.0, 21 and 48 mg/L TWA concentrations, respectively.

Statistically significant inhibition of growth rate was found at TWA concentrations of 7.0 mg/L and higher, however, the effect observed at 7.0 mg/L was considered biologically insignificant (<10%). Therefore this value is used as NOEC. In none of the test concentrations inhibition of growth rate >= 50% was reached. The ErC10 and ErC50 based on TWA concentrations were 8.8 and 58 mg/L, respectively.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed for 72 hours to 5 concentrations of the test substance, prepared from a saturated solution of the test material prepared at 100 mg/L (three replicate flasks per concentration), under constant illumination and shaking at a temperature of 22 - 24°C. The test material solutions were prepared by magnetic stirring at room temperature for 70 - 88 minutes followed by siphoning off the aqueous phase to exclude undissolved material. 
Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. Measured concentrations at test initiation ranged from 89 - 101% of nominal, and decreased to 78 - 88% of nominal after 24 hours and 0.07 - 10% of nominal at test end (mean of 2 measurements). Therefore time-weighted average (TWA) concentrations were calculated and used for expression of endpoints. Nominal concentrations of 10, 18, 32, 56 and 100% of the saturated solution prepared at 100 mg/L corresponded with 3.3, 7.0, 11, 8.0, 21 and 48 mg/L TWA concentrations, respectively.
Statistically significant inhibition of growth rate was found at TWA concentrations of 7.0 mg/L and higher, however, the effect observed at 7.0 mg/L was considered biologically insignificant (<10%). Therefore this value is used as NOEC. In none of the test concentrations inhibition of growth rate >= 50% was reached. The ErC10 and ErC50 based on TWA concentrations were 8.8 and 58 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
58 mg/L
EC10 or NOEC for freshwater algae:
8.8 mg/L

Additional information