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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:
03 Nov 2017 - 11 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations.
- Sampling method: 1.8 mL, at t=0 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- 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 an intermediate item concentration but without algae and samples for analysis were taken at the start and at the end of the test period.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium. Test solutions were prepared at the highest concentration of 100 mg/L applying a 54-64 minute period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration 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.

Test organisms

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
- 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.
- Pre-culture: 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. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505).
- Pre-culture medium and test medium: M2, according to OECD 201.

Study design

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

Test conditions

Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22 - 23 °C
pH:
At t=0 h: 8.1-8.4
At t=72 h: 7.6-7.7
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentrations were 102-120% of nominal throughout the test (averaged for both anionic and cationic concentrations). Therefore, effect parameters were based on nominal concentrations. See Table 1 and Table 2 in 'Any other information on results incl. tables' for details on measured concentrations throughout the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean): 241 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium, formulated using Milli-RO water (tap-water purified by reverse osmosis).
- Intervals of water quality measurement: pH: at the beginning and at the end of the test. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 92 to 94 μE/m^2/s

EFFECT PARAMETERS MEASURED
- Cell density was measured at t= 24, 48, and 72 h.
- 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 (pathlength = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Combined Limit/Range-Finding Test concentrations: Control, 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected ErC50 was between 10.0 and 100 mg/L (nominal concentration).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Sep 2017)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 15-22 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 9.5-11 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological significance
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 10 mg/L when compared to the control.
- Growth rates were in the range of the controls at the lowest test concentration. At nominal concentrations of 3.2 mg/L and upwards, inhibition of growth rates increased with increasing concentration. Statistically significant inhibition of growth rate was found at test concentrations of 3.2 mg/L and higher. The effects observed at 3.2 and 10 mg/L, however, were below 10% and therefore considered to be biologically not relevant. The NOEC for growth rate inhibition was thus set at 10 mg/L.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. 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:
Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
ECx: Weibit analysis using linear max. likelihood regression.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Final Test: Test Samples - Anion

Time of sampling
[hours]

Test item concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

n.d.

n.a.

 

 

1.0

1.19

119

 

 

3.2

3.63

113

 

 

10

10.9

109

 

 

10 (a)

11 (b)

112

 

 

32

33.9

106

 

 

100

103

103

 

72

0

n.d.

n.a.

n.a.

 

1

1.22

122

102

 

3.2

3.71

116

102

 

10

11 (b)

111

102

 

10 (a)

12 (b)

121

108

 

32

36.8

115

109

 

100

103

103

101

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

(a) Without algae.

(b)  Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 2: Final Test: Test Samples - Cation

Time of sampling
[hours]

Test item concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

n.d.

n.a.

 

 

1.0

1.13

113

 

 

3.2

3.59

112

 

 

10

11.1

111

 

 

10 (a)

10.9

109

 

 

32

34.3

107

 

 

100

101

101

 

72

0

n.d.

n.a.

n.a.

 

1.0

1.15

115

102

 

3.2

3.62

113

101

 

10

10.9

109

99

 

10 (a)

12 (b)

117

107

 

32

35.1

110

103

 

100

102

102

101

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

(a) Without algae.

(b) Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 3: Growth Rate And Percentage Inhibition For The Total Test Period 

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.828

0.0245

6

1.0

1.806

0.0043

3

1.2

3.2

1.760

0.0113

3

3.7*#

10

1.664

0.0365

3

9.0*#

32

0.000

0.0000

3

100*

100

0.000

0.0000

3

100*

* effect was statistically significant;

#effect biologically not relevant (<10%)

 

Table 4: Growth Rate And Percentage Inhibition At Different Time Intervals 

Nominal conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.883

 

2.049

 

1.553

 

1.0

3

1.852

1.6

2.024

1.2

1.543

0.63

3.2

3

1.747

7.2

2.020

1.4

1.515

2.5

10

3

1.596

15

2.027

1.1

1.369

12

32

3

0.000

100

0.000

100

0.000

100

100

3

0.000

100

0.000

100

0.000

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria
Conclusions:
The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 18 mg/L, 10 mg/L and 10 mg/L (based on biological relevance), based on nominal concentrations.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at nominal concentrations

of 1.0, 3.2, 10, 32 and 100 mg/L (3 replicates) and an untreated control (6 replicates). Measured concentrations in the test solution were at 102 -120% relative to nominal throughout the test (averaged over both anionic and cationic parts of the test substance). Therefore, effect parameters were based on nominal concentrations. The EC50 for growth rate inhibition (72h-ErC50) was 18 mg/L, the 72h-ErC10 was 10 mg/L. The 72h-NOErC was 1.0 and 10 mg/L based on statistical significance and biological relevance, respectively. The study met all validity criteria, and is considered reliable without restriction.