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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 2017 - 8 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
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
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008 (amended by EC No. 2016/266 )
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Solubility in water: 86 mg/L at 24°C
- Vapour pressure: 0.60 Pa at 24°C
Analytical monitoring:
yes
Details on sampling:
Single samples for analysis were taken from all test concentrations and the control.
Frequency at t=0 h, t=24 h and t=72 h
Volume 3.0 mL
Storage Samples were stored in a freezer (≤-15ºC) until analysis.

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 an additional test vessel at 10% (v/v) of the saturated solution prepared at a loading rate of 100 mg/L, but without algae, and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of a saturated solution (direct addition to test medium) - A loading rate of 100 mg/L was stirred overnight on a magnetic stirring device to accelerate the dissolution of the test item in medium. The obtained mixture was allowed to settle for 30 minutes. The resulting aqueous Saturated Solution (SS) was siphoned off and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 50 mL 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.

- Controls: test medium without test item or other additives

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
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
- Age of inoculum (at test initiation): 3 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.
- 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. Stock culture medium = M1 (according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)

ACCLIMATION
- Acclimation period: yes (3 days)
- Culturing media and conditions (same as test or not): yes, pre-culture medium and test medium are the same (M2, according to OECD 201), as are pre-culture and test conditions
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L (as CaCO3)
Test temperature:
22-23°C
pH:
8.0-8.1
Nominal and measured concentrations:
Nominal test concentrations: 1.0, 3.2, 10, 32 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L.

At the start of the test, the actual test concentrations were in agreement with the nominal concentrations, i.e., were 0.87, 2.8, 8.6, 27.1 and 89.1 mg/L in 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution.
At the end of the test, these measured concentrations had decreased to 15-53% of the initial concentrations; i.e. 0.13, 1.02, 4.13, 14.4 and 31.0 mg/L in 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution.

Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated and used to determine the effect parameters and these were: 0.43, 1.7, 6.0, 18 and 51 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped
- Fill volume: 50 mL
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 93.2 x 10^4 cells/mL (mean, n=6)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 or 2 replicates of each test concentration without algae. 1 extra replicate of each test concentration for sampling purposes after 24 hours of exposure.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature - continuously in a temperature control vessel; pH - at the beginning and at the end of the test for all test concentrations and the control

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: TLD-lamps with a light intensity within the range of 84 to 87 µE/m^2/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- 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 cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2

RANGE FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected EC50 for both growth rate inhibition and yield inhibition was between 10% and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (February 2017)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% Confidence interval is: 7.4-13 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 g/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% Confidence interval is: 5.3-6.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.7 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: no - Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 6.0 mg/L when compared to the control.
- Any stimulation of growth found in any treatment: yes - at the two lowest test concentrations
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Other: After 24 hours, the measured concentrations had decreased to 63-79% of the initial concentrations. At the end of the test, the measured concentrations had decreased to 15-53% of the initial concentrations. The concentrations measured in the samples without algae were comparable, thus it is unlikely that the substance was lost due to sorption by the algal biomass.
- Effect concentrations exceeding solubility of substance in test medium: no

pH and temperature were within the limits prescribed in the OECD guideline.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h-ErC50: 1.2 mg/L (95% C.I. 1.1-1.2 mg/L)
- Other: The ErC50 value for the positive control fell into the historical ranges for growth rate inhibition (between 0.82 and 2.3 mg/L).
Reported statistics and error estimates:
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).

Since stimulation rather than inhibition of growth rate was observed at the two lowest concentrations and complete inhibition at the two highest test concentrations, the lowest and the highest test group were removed from the statistical determination of the NOEC.
Due to mathematical reasons (The requirements for the parametric test (i.e. normality of data and homogeneity of variance) were not fulfilled and the replication was not sufficient for a non-parametric test), statistical analysis would otherwise not have been possible to be performed for the data gathered after 72 hours of exposure to determine the NOEC.
The EC10 was still calculated as well as an equivalent alternative.

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA exposure concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Final Test: Test Samples

Time of sampling
[hours]

Percentage of SS1
[%]

Analyzed concentration2
[mg/L]

Relative to initial
[%]

0

0

< LOQ

 

 

1.0

0.8724

 

 

3.2

2.83

 

 

10

8.57

 

 

103

8.71

 

 

32

27.1

 

 

100

89.1

 

24

0

< LOQ

n.a.

 

1.0

0.6034

69

 

3.2

1.93

68

 

10

6.62

77

 

103

6.91

79

 

32

17.0

63

 

100

55.9

63

72

0

< LOQ

n.a.

 

1.0

0.1314

15

 

3.2

1.024

36

 

10

4.13

48

 

103

4.04

46

 

32

14.4

53

 

100

31.0

35

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

2              Samples were stored in the freezer (≤ -15°C) until the day of analysis.

3              Without algae.

4              Content calculated after correction of the test item response with the mean peak area of the background of the blank QC samples.

< LOQ   Below the limit of quantification (LOQ) at 0.1 mg/L.

n.a.        Not applicable.

Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated and used to determine the effect parameters.

Measured concentrations versus nominal concentrations

Hyacinth Body # 3
% SS at 100 mg/L

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72 h

1.0

0.872

0.603

0.131

0.43

3.2

2.83

1.93

1.02

1.7

10

8.57

6.62

4.13

6.0

101

8.71

6.91

4.04

6.1

32

27.1

17.0

14.4

18

100

89.1

55.9

31.0

51

1without algae

Biological results

A stimulation of growth rates was observed at the two lowest test concentrations during the 72-hour test period, whereas the growth rate of algae exposed to TWA concentrations of 6.0 mg/L and higher were increasingly reduced.

Statistically significant inhibition of growth rate and yield was found at test concentrations of 6.0 mg/L and higher.

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

Hyacinth Body # 3
TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.510

0.0334

6

 

0.43

1.565

0.0212

3

-3.61

1.7

1.576

0.0242

3

-4.4

6.0

1.320

0.1017

3

12.6*

18

0.008

0.0136

3

99.5*

51

0.012

0.0209

3

99.21

1Test group not used in statistical determination of the NOEC.

* effect was statistically significant

Percentage inhibition of growth rate at different time intervals during the final test

Hyacinth Body # 3
TWA conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.032

 

1.878

 

1.620

 

0.43

3

1.288

-24.8

1.788

4.8

1.619

0.0

1.7

3

1.558

-51.0

1.543

17.8

1.628

-0.5

6.0

3

0.757

26.7

1.637

12.9

1.567

3.3

18

3

0.627

39.2

-0.465

124.8

-0.139

108.6

51

3

0.012

98.8

0.187

90.0

-0.163

110.1

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks' field
Conclusions:
The ErC50, ErC10 and NOEC were 8.4, 5.8 and 1.7 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was also performed under GLP and assigned a Klimisch 1 rating. Test solutions were prepared at a loading rate of 100 mg/L, resulting in an aqueous Saturated Solution (SS) that was used as the highest test concentration. Lower test concentrations (1.0, 3.2, 10 and 32% (v/v) of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Concentrations were analyzed at t = 0, 24 and 72 h. The initial measured concentration in the 100% saturated solution was 89% of nominal, and in dilutions from the 100% saturated solution in the range 85 -88% of nominal. At the end of the test, the measured concentrations had decreased to 15-53% of the initial concentrations. The concentrations measured in the samples without algae were comparable, thus it is unlikely that the substance was lost due to sorption by the algal biomass. Since test concentrations gradually decreased during the test period, time-weighted average measured concentrations were calculated: 0.43, 1.7, 6.0, 18 and 51 mg/L, corresponding with 1.0%, 3.2%, 10%, 32% (v/v) and 100% of the SS prepared at a loading rate of 100 mg/L. Statistically significant inhibition of growth rate (and yield) was found at test concentrations of 6.0 mg/L and higher. The ErC50, ErC10 and NOEC were 8.4, 5.8 and 1.7 mg/L, respectively, based on time-weighted average measured concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The result derived from read across
Justification for type of information:
The read across justification for Daphnia and algae is presented in the Endpoint summary for Aquatic toxicity and the accompanying files are also attached there.
Reason / purpose for cross-reference:
read-across source
Water media type:
freshwater
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% Confidence interval is: 7.4-13 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 g/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% Confidence interval is: 5.3-6.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Remarks:
adequate and reliable documentation on the read across is provided
Conclusions:
Based on the results of the study for read-across substance Hyacinth body #3, the ErC50, ErC10 and NOEC for Hyacinth body were 8.4, 5.8 and 1.7 mg/L, respectively.

Description of key information

The ErC50, ErC10 and NOEC are 8.4, 5.8 and 1.7 mg/L, respectively, based on read-across from Hyacinth body #3.

Key value for chemical safety assessment

EC50 for freshwater algae:
8.4 mg/L
EC10 or NOEC for freshwater algae:
5.8 mg/L

Additional information

For Hyacinth body read across from Hyacinth body #3 was applied. Read-across justification is added in the endpoint summary of Aquatic toxicity because also the Daphnia toxicity is presented there. Below the experimental results for Hyacinth body #3 are presented.

Algae toxicity study with Hyacinth body #3

A study with Hyacinth body #3 was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was also performed under GLP and assigned a Klimisch 1 rating. Test solutions were prepared at a loading rate of 100 mg/L, resulting in an aqueous Saturated Solution (SS) that was used as the highest test concentration. Lower test concentrations (1.0, 3.2, 10 and 32% (v/v) of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Concentrations were analyzed at t = 0, 24 and 72 h. The initial measured concentration in the 100% saturated solution was 89% of nominal, and in dilutions from the 100% saturated solution in the range 85 -88% of nominal. At the end of the test, the measured concentrations had decreased to 15-53% of the initial concentrations. The concentrations measured in the samples without algae were comparable, thus it is unlikely that the substance was lost due to sorption by the algal biomass. Since test concentrations gradually decreased during the test period, time-weighted average measured concentrations were calculated: 0.43, 1.7, 6.0, 18 and 51 mg/L, corresponding with 1.0%, 3.2%, 10%, 32% (v/v) and 100% of the SS prepared at a loading rate of 100 mg/L. Statistically significant inhibition of growth rate (and yield) was found at test concentrations of 6.0 mg/L and higher. The ErC50, ErC10 and NOEC were 8.4, 5.8 and 1.7 mg/L, respectively, based on time-weighted average measured concentrations.