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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:
18 Jul 2018 - 22 Aug 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:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006, Annex 5 corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white powder
Storage conditions: at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: 1.9 mL from the approximate centre of the test vessels 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 the WAF prepared at a loading rate of 32 mg/L 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 of substance to test medium.
Since the test item consisted of a mixture of two enantiomers, and because the test item was not completely soluble in test medium, WAFs were prepared individually at loading rates ranging between 1.0 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous WAFs were collected by filtration through a 0.45 μm membrane filter (PES450, Supor®) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
- 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: Raphidocelis subcapitata (formerly known as 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.
- Stock culture medium: M1 medium, according to NPR 6505
- Pre-culture: 3-4 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.
- Pre-culture medium and test medium: M2 medium, 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 - 24°C
pH:
At start (t=0 h): 8.0-8.4
At end (t=72 h): 8.0 - 8.2
Nominal and measured concentrations:
Nominal: control and WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
Measured: The measured concentrations were at the level of nominal (i.e. 90 – 105% relative to nominal) throughout the test. Effect parameters were therefore expressed in terms of analytically confirmed nominal concentrations. See Table 1 in 'Any other information on results' for details on measured concentrations.
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: 10^4 cells/mL
- Control end cells density: 158 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additional replicates: 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, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations and the control. 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 83 - 87 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
- 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 WAF prepared at 56 mg/L and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: control and WAFs individually prepared at 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Jul 2018)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 58-69%
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on statistical significance
Details on results:
- A well-defined EC50 for growth rate inhibition could not be determined because the observed effects were below 50%.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 56 mg/L when compared to the control.
- No significant inhibition of growth rate and reduction of yield was recorded at the three lowest test concentrations, while at the highest test concentration, an inhibition of 30 and 78% for growth rate and yield, respectively, was observed at the end of the test. Statistically significant inhibition of both growth rate inhibition and yield was found at the two highest test concentrations. However, since effects <10% were considered as biologically not relevant, the NOEC for growth rate inhibition was set at 56 mg/L based on biological relevance.
- Analytical results: The measured concentrations were at the level of nominal (i.e. 90 – 105% relative to nominal) throughout the test. Therefore, it can be stated that the undissolved material removed during the preparation of test solutions was most likely not test item related. It should be noted that a small response was measured in the samples taken from the control, however, was considered to be negligible and did not impact the results of the study. The maximum contribution to the samples at the lowest concentration level was 0.26%. The concentrations measured in the samples without algae were comparable to the concentrations measured in the samples with algae.
- 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 0.90 mg/L with a 95% confidence interval ranging from 0.88 - 0.93 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 calculation: 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis with the percentages of growth rate inhibition versus the logarithms of the corresponding test item concentrations.
- 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: Final Test: Test Samples

Time of sampling
[hours]

Loading rate(a)

[mg/L]

Concentration
analyzed
[mg/L]

Relative to
initial
[%]

0

0

     0.0043(c)

 

 

10

9.62

 

 

18

16.3

 

 

32

32.3

 

 

 32(b)

32.6

 

 

56

53.6

 

 

100

102

 

72

0

   0.029(c)

n.a.

 

10

10.2

106

 

18

16.3

100

 

32

32.5

101

 

 32(b)

32.7

100

 

56

55.1

103

 

100

105

103

(a) A water accommodated fraction (WAF) prepared at the loading rate.

(b) Without algae.

(c)  Estimated value, calculated by extrapolation of the calibration curve. The maximum contribution to the samples at the lowest concentration level was 0.26%

n.a. Not applicable.

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


Nominal conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.686

0.0334

6

 

10

1.685

0.0205

3

0.016

18

1.690

0.0179

3

-0.27

32

1.668

0.0402

3

1.1

56

1.568

0.0325

3

7.0#

100

1.187

0.0373

3

30*

* - effect was statistically significant

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

Table 3: 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

1.755

 

1.703

 

1.599

 

10

3

1.739

0.89

1.781

-4.6

1.536

3.9

18

3

1.879

-7.1

1.735

-1.9

1.457

8.9

32

3

1.887

-7.5

1.697

0.34

1.420

11

56

3

1.855

-5.7

1.476

13

1.373

14

100

3

1.749

0.33

0.513

70

1.298

19

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 72h-EC50 of the substance for growth rate inhibition (ErC50) was > 100 mg/L. The 72h-ErC10 was 63 mg/L (95% confidence interval: 58 - 69 mg/L), based on analytically confirmed nominal concentrations. The 72h-NOEC was 32 mg/L based on statistical significance and 56 mg/L based on biological relevance.
Executive summary:

In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Water Accommodated Fractions of the substance, individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L (3 replicates each) and an untreated control (6 replicates).

Measured concentrations of the substance were at the level of nominal (i.e. 90 - 105% relative to nominal) throughout the test, and effect parameters were therefore based on analytically confirmed nominal concentrations.

No significant inhibition of growth rate was recorded at the three lowest test concentrations, while growth rates of algae exposed to loading rates of 56 and 100 mg/L were inhibited by 7.0 and 30%, respectively, at the end of the test.

The 72h-EC50 for growth rate inhibition (ErC50) was > 100 mg/L. The 72h-ErC10 was 63 mg/L (95% confidence interval: 58 - 69 mg/L). The 72h-NOEC for growth rate inhibition was 32 mg/L based on statistical significance and 56 mg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.