Registration Dossier

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 September 2018 - 05 November 2018
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:
2019

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:
liquid: viscous
Details on test material:
- Appearance: Brown viscous liquid
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L and control
- Sampling method: 1.8 mL from the approximate centre of the test vessels, at t=0 h, t=24 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 nominally 3.2 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to test medium. The test solution with highest concentration of 100 mg/L (corrected for purity) was prepared by applying one day 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:
21 - 22°C
pH:
At t=0 h: 7.3 - 8.2
At t=72 h: 7.9 - 8.3
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentrations: initial concentrations were 0.31, 0.85, 2.3, 7.9, 25 and 91 mg/L (72-96% of nominal concentrations) which remained stable, i.e. were at the level of 85 – 113%, throughout the test. Therefore the initially measured concentrations were used to determined effect parameters.
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: 1 x 10^4 cells/mL
- Control end cells density: 232 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 extra replicates of each test group for sampling purposes after 24 hours of exposure, 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 according to OECD 201, formulated using water purified by
reverse osmosis.
- Intervals of water quality measurement: pH: at beginning and end of test; temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 70 to 78 μE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Cell density was measured at t=24 h, t=48 h and t=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 (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the nominal concentrations of 3.2 mg/L and 10 mg/L to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Test concentrations in Combined Limit/Range-Finding Test: 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes, ErC50 is estimated to be between 10 and 100 mg/L (nominal concentrations).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Nov 2018)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 16 - 18 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 2.5 - 2.9 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.85 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.31 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical 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 initially measured concentrations of 2.3 and 7.9 mg/L when compared to the control.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- Growth rate inhibition increased with increasing concentrations of test item at all test concentrations, resulting in 85% growth rate inhibition reduction at the highest test concentration. Statistically significant growth rate inhibition was found at all test concentrations. However, growth rate inhibition recorded at the lowest two test concentrations was below 10% and therefore considered to be biologically not relevant. The NOEC based on biological relevance was thus set at 0.85 mg/L.
- The concentrations measured in the solutions incubated with algae were comparable to those measured in their counterparts incubated without algae throughout the test. Therefore, it was concluded that the presence of the algae had no effect on the concentrations of the test item in test
medium.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 for growth rate inhibition (72h-ERC50) was 1.05 mg/L with a 95% confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 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, 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 reduction 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.

Any other information on results incl. tables

Table 1: Measured Concentrations in test samples

Time of sampling
[hours]

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

n.d.

n.a.

 

 

0.32

0.307

96

 

 

1.0

0.845

84

 

 

3.2

2.30

72

 

 

3.2 1

2.53

79

 

 

10

7.85

78

 

 

32

25.0

78

 

 

100

90.8

91

 

24

0

n.d.

n.a.

n.a.

 

0.32

0.288

90

94

 

1.0

0.803

80

95

 

3.2

2.60

81

113

 

3.2 1

2.57

80

101

 

10

6.36

64

81

 

32

26.3

82

105

 

100

82.3

82

91

72

0

0.0071 2

n.a.

n.a.

 

0.32

0.246

77

85

 

1.0

0.834

83

104

 

3.2

2.67

83

103

 

3.2 1

2.20

69

86

 

10

7.18

72

113

 

32

26.5

83

101

 

100

86.4

86

105

1     Without algae.

2      Estimated value, calculated by extrapolation of the calibration curve. The maximum contribution to 0.32 mg /L test sample is 3.4%.

n.d. Not detected.

n.a. Not applicable.

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

Initially measured conc. (mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.815

0.0152

6

 

0.31

1.778

0.0121

3

2.1#

0.85

1.754

0.0123

3

3.4#

2.3

1.642

0.0308

3

9.5*

7.9

1.353

0.0235

3

25*

25

0.637

0.0557

3

65*

91

0.275

0.0190

3

85*

* effect was statistically significant,#effect was statistically significant however biologically not relevant (<10%).

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

Initially measured conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.021

 

1.938

 

1.486

 

0.31

3

2.087

-3.3

1.835

5.3

1.410

5.1

0.85

3

2.064

-2.1

1.799

7.2

1.398

6.0

2.3

3

2.004

0.85

1.571

19

1.352

9.0

7.9

3

1.861

7.9

1.072

45

1.125

24

25

3

1.461

28

0.484

75

-0.035

102

91

3

0.920

54

0.025

99

-0.121

108

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' section for details on validity criteria.
Conclusions:
The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 17 mg/L, 2.7 mg/L and 0.85 mg/L (based on biological relevance), based on initially measured concentrations (corrected for purity).