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Ecotoxicological information

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:
2016-08-04 to 2016-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2011)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2016, Commission Regulation (EU) No 2016/266)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-12-16
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations, with exception of the two lowest concentrations (1:100 and 1:320), were analysed at test start and at test end.
- Sampling method: duplicate samples of all concentrations
- Sample storage conditions before analysis: samples were stored at about 20 °C until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: before sample weight the test item was melted at about 65 °C in a water bath. Afterwards, 200.2 mg test item was dispersed in 2000 mL test water, followed by 15 minutes ultrasonic treatment and 96 hours of intensive stirring at room temperature in the dark. After stirring, the suspension was filtered (0.45 µm membrane filter Whatman, NC45).
- Controls: yes, test water only
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source: cultivated at the laboratory
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: same as test conditions

ACCLIMATION
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
23 °C
pH:
7.2 - 7.3
Nominal and measured concentrations:
Nominal concentrations: Control, Dilution 1:320, 1:100, 1:32, 1:10, 1:3.2, Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L
Arithmetic mean measured concentration:
Details on test conditions:
TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flask filled with 30 mL
- Type: coverd with glass lid
- Initial cells density: 5000 cells/mL
- Control end cells density: 72.9 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water

OTHER TEST CONDITIONS
- Photoperiod: 24 h/d light
- Light intensity and quality: 68 µE / m2 • s (range 66 to 70 µE / m2 • s)


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (Cell Counter CASY TT, Germany)
- Other: shape and size were visulally inspected

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: Undiluted filtrate, Dilution 1:10 and 1:100
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
39 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-CI: 35 - 45 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-CI: 15 - 20 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-CI: 16 - 20 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-CI: 2.5 - 5.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.0 mg/L after 72 hours (July 2016)
Reported statistics and error estimates:
The 72-hour EC values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Weibull Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate. Statistical analysis was performed using ToxRat Professional.

Calculated arithmetic mean measured concentrations

Treatment /
Dilution

 

 

Analytical Measured Concentration
of the Test Item
[mg/L]

Mean Measured Concentration (Arithmetic Mean)

[mg/L]

0 hours

72 hours

Dilution 1:320

n.a.

n.a.

n.a.

Dilution 1:100

n.a.

n.a.

n.a.

Dilution 1:32

2.42

2.36

2.4

Dilution 1:10

8.22

8.71

8.5

Dilution 1:3.2

26.1

26.5

26

Undiluted Filtrate°

82.6

82.9

83

°: Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

n.a.: Not analyzed since below NOEC determined in this test

Rounded biomass results obtained by calculation using the exact raw data

Treatment/Dilution

Mean Measured Concentration

(mg/L)

Repl.

No.

Biomass of Algae*

24 hours

48 hours

72 hours

Control

1

2

3

4

5

6

2.8

2.5

2.0

2.0

3.0

2.5

13.8

15.4

14.5

13.1

13.0

15.4

77.0

68.9

76.6

70.0

69.8

75.2

Mean

SD

2.5

0.4

14.2

1.1

72.9

3.7

1:320

n.a.

1

2

3

2.5

2.5

2.5

13.9

13.7

13.7

72.4

68.9

71.1

Mean

SD

2.5

0.0

13.8

0.1

70.8

1.7

1:100

n.a.

1

2

3

2.4

2.0

2.4

14.5

13.7

15.9

77.6

69.9

78.6

Mean

SD

2.3

0.2

14.7

1.1

75.4

4.7

1:32

2.4

1

2

3

3.1

2.9

1.6

12.2

12.8

12.0

73.4

68.0

68.6

Mean

SD

2.5

0.8

12.4

0.4

70.0

3.0

1:10

8.5

1

2

3

1.7

3.1

1.6

12.3

11.2

13.1

58.2

53.9

53.3

Mean

SD

2.2

0.8

12.2

0.9

55.1

2.7

1:3.2

26

1

2

3

1.8

1.7

1.6

8.4

6.2

7.2

26.6

22.3

23.9

Mean

SD

1.7

0.1

7.3

1.1

24.3

2.2

Undiluted

Filtrate°

83

1

2

3

0.3

0.3

0.5

0.6

0.4

0.5

0.4

0.4

0.4

Mean

SD

0.4

0.12

0.5

0.08

0.4

0.05

°: Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units

(x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 1.0 x 104relative fluorescence units.

 

Average Growth Rates (µ)

Treatment/

Dilution

Mean Measured Concentration

(mg/L)

Average Growth Rate µ (day-1) and Inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

1:320

1:100

1:32

1:10

1:3.2

Undilut. Filtrate°

--

n.a.

n.a

2.4

8.5

26

83

1.558

1.586

1.489

1.559

1.390

1.219#

-0.282#

0.0

-1.8

4.5

-0.1

10.8

21.8

118.1

1.659

1.646

1.678

1.591*

1.584*

1.322*

-0.037*

0.0

0.8

-1.1

4.1

4.5

20.3

102.2

1.652

1.643

1.663

1.639

1.559#

1.285#

-0.078#

0.0

0.6

-0.7

0.8

5.6

22.2

104.7

°: Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller,α= 0.05).

#: Mean value statistically significantly lower than in the control (according to a Welch t-test one-sided smaller,α= 0.05).

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Yield (Y) 

Treatment/

Dilution

Mean Measured Concentration

(mg/L)

Yield Y (x 104) and Inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Control

1:320

1:100

1:32

1:10

1:3.2

Undilut. Filtrate°

--

n.a.

n.a.

2.4

8.5

26

83

1.9

2.0

1.8

2.0

1.6

1.2*

-0.1*

0.0

-2.3

9.4

-3.4

15.9

37.2

105.9

13.7

13.3

14.2

11.8*

11.7*

6.8*

0.0*

0.0

2.9

-3.8

13.4

14.5

50.6

100.2

72.4

70.3

74.9

69.5

54.6*

23.8*

-0.1*

0.0

2.9

-3.4

4.0

24.6

67.2

100.1

°: Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller,α= 0.05).

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Section-by-Section Growth Rates

Treatment/

Dilution

Mean Measured Concentration

(mg/L)

Section-by-Section Growth Rates (day-1) and

inhibition of Growth Rates (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

1:320

1:100

1:32

1:10

1:3.2

Undilut. Filtrate°

---

n.a.

n.a.

2.4

8.5

26

83

1.558

1.586

1.489

1.559

1.390

1.219

-0.282

0.0

-1.8

4.5

-0.1

10.8

21.8

118.1

1.761

1.706

1.867

1.623

1.779

1.425

0.208

0.0

3.1

-6.0

7.8

-1.0

19.1

88.2

1.638

1.636

1.634

1.734

1.508

1.211

-0.161

0.0

0.1

0.2

-5.9

7.9

26.0

109.8

°: Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Analytical Results

Sampling

Day / Age of Sample

(d/h)

Treatment/

Dilution

Measured

Concentration

of Test Item

x

(mg/L)

Sample

Preparation

Factor

F

Determined

Concentration

of Test Item

c

(mg/L)

% of Initially Measured Concentration

 

(%)

0/0

(fresh)

Control

1:32

1:10

1:3:2

Undiluted Filtrate°

n.d.

8.07

27.4

87.1

275

0.3

0.3

0.3

0.3

0.3

<LOQ

2.42

8.22

26.1

82.6

 

3/72

(aged)

Control

1:32

1:10

1:3:2

Undiluted Filtrate°

n.d.

7.87

29.0

88.4

276

0.3

0.3

0.3

0.3

0.3

<LOQ

2.36

8.71

26.5

82.9

n.a.

98

106

102

100

LOQ = 0.15 mg test item /L

n.a. = not applicable

° = undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:
yes
Conclusions:
The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined as follows:
72h-ErC10 (growth rate) = 18 mg/L
72h-ErC50 (growth rate) = 39 mg/L
NOEC (growth rate) = 2.4 mg/L
All effect concentrations are based on arithmetic mean measured concentrations of the test item. However, the recovery rates are within the range of 80-120 %.
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266 C3, 2016. Due to the low water solubility (<100 mg/L) of the test item in reconstituted test water (AAP Medium), the test media were prepared following the filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 24 hours. After stirring the suspension was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320 of the filtrate were used as test media. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The analytical measurements from test media at the start of the test showed the correct preparation of the test media, i.e. the theoretical spacing factor of about 3.2 between the different test concentrations was met. At the end of the test, 98 to 106 % of the initially measured concentrations were found. Thus, the test item was stable over the test period of 72 hours. Therefore, mean measured concentrations were calculated as the arithmetic mean of the concentrations measured at the start and the end of the test. The 72-hour ErC10 (growth rate) was determined to be 18 mg/L with a 95 % convidence interval of 15 and 20 mg/L and the 72-hour ErC50 (growth rate) resulted in 39 mg/L (95 %-CI: 35 - 45 mg/L).

Description of key information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined as follows:

72h-ErC10 (growth rate) =  18 mg/L

72h-ErC50 (growth rate) = 39 mg/L

NOEC (growth rate) =  2.4 mg/L

All effect concentrations are based on arithmetic mean measured concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:
39 mg/L
EC10 or NOEC for freshwater algae:
18 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266 C3, 2016. Due to the low water solubility (<100 mg/L) of the test item in reconstituted test water (AAP Medium), the test media were prepared following the filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 24 hours. After stirring the suspension was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320 of the filtrate were used as test media. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The analytical measurements from test media at the start of the test showed the correct preparation of the test media, i.e. the theoretical spacing factor of about 3.2 between the different test concentrations was met. At the end of the test, 98 to 106 % of the initially measured concentrations were found. Thus, the test item was stable over the test period of 72 hours. Therefore, mean measured concentrations were calculated as the arithmetic mean of the concentrations measured at the start and the end of the test. The 72-hour ErC10 (growth rate) was determined to be 18 mg/L with a 95 % convidence interval of 15 and 20 mg/L and the 72-hour ErC50 (growth rate) resulted in 39 mg/L (95 %-CI: 35 - 45 mg/L).