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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:
12 Jan 2018 - 22 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
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
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:
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

Constituent 1
Chemical structure
Reference substance name:
(8R,9S,10R,13S,14S,17R)-17-ethynyl-17-hydroxy-13-methyl-1,2,8,9,10,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one
EC Number:
608-630-3
Cas Number:
31528-46-8
Molecular formula:
C20H24O2
IUPAC Name:
(8R,9S,10R,13S,14S,17R)-17-ethynyl-17-hydroxy-13-methyl-1,2,8,9,10,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Off-white powder
Test item storage: At room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations
- Sampling method: 2.0 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 the limit concentration (i.e. the undiluted SS prepared at 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a nominal loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) 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

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: Three days before the start of the test, cells from the algal stock culture were inoculated in pre-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:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21 - 23 °C
pH:
At t=0 h: 8.1
At t=72 h: 8.3-8.4
Nominal and measured concentrations:
Nominal concentrations: untreated control, 1.0, 10 and 100% of a saturated solution prepared at a nominal loading rate of 100 mg/L
Initially measured concentration in undiluted satured solution (100% of SS): 1.21 mg/L.
Since the measured concentration in undiluted satured solution remained stable throughout the test (99% of initial), effect parameters were based on initial measured concentrations.
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 cell density: 1 x 10^4 cells/mL
- Control end density (mean): 300 x 10^4 cells/mL
- 3 replicates of each 1.0 and 10% of saturated solution
- 6 replicates of the control and undiluted saturated solution
- 1 extra replicate 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, 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 control and undiluted SS. Temperature: continuously in a temperature control vessel.
- Appearance of the cells: at the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae compared to the control.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity of 86 µE/m^2/s

EFFECT PARAMETERS MEASURED
- Cell density at t=24, 48 and 72 h in the control and limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
- 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). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Jan 2018)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: The measured concentration of 1.2 mg/L is considered to reflect the maximum soluble amount of the test item in test medium at a loading rate of 100 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: The measured concentration of 1.2 mg/L is considered to reflect the maximum soluble amount of the test item in test medium at a loading rate of 100 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Based on biological relevance
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 the undiluted SS when compared to the control.
- 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
- EC50 for growth rate inhibition (72h-ErC50) was 0.86 mg/L (95% confidence interval 0.84 - 0.88 mg/L). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Statistical significance: Two-Sample T-Test, α=0.05, one-sided, smaller
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Test Samples

Time of sampling
[hous]

Date of sampling

Date of
analysis (a)

Percentage of SS (b)
[%]

Analyzed concentration
[mg/L]

Relative to
initial
[%]

0

15 Jan 2018

22 Jan 2018

0

n.d.

 

 

 

 

100

1.21

 

 

 

 

 100 (c)

1.27

 

24

16 Jan 2018

22 Jan 2018

0

n.d.

n.a.

 

 

 

100

1.22

101

 

 

 

 100 (c)

1.29

102

72

18 Jan 2018

22 Jan 2018

0

0.000083 (d)

n.a.

 

 

 

100

1.20

99

 

 

 

 100 (c)

1.19

94

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

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

(c) Without algae.

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

n.a. Not applicable.

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

Test item
%SS prep. at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.901

0.0281

6

 

1.0

1.896

0.0287

3

0.251

10

1.871

0.0072

3

1.51

100

1.746

0.0148

6

8.1 *#

* effect was statistically significant;#effect biologically not relevant (<10%);
1effect was statistically not compared against the control.

Table 3: Growth Rate and Percentage Inhibition at different time intervals

Test item
%SS prep. at 100 mg/L

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.341

 

1.764

 

1.597

 

10

6

2.439

-4.2

1.473

16

1.326

17

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
No biologically relevant inhibition of growth rate was recorded at any of the concentrations tested. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded a measured concentration of 1.2 mg/L being considered the maximum soluble amount of the test item in test medium at a nominal loading rate of 100 mg/L. The 72h-ErC10 was >1.2 mg/L. The 72h-NOEC for growth rate inhibition was 1.2 mg/L based on biological relevance and <1.2 mg/L based on statistical significance.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 1.0, 10 and 100% of saturated solutions prepared at a nominal loading rate of 100 mg/L and an untreated control (triplicates for 1.0 and 10% of SS, 6 replicates for control and 100% of SS). Measured concentrations in the undiluted SS remained stable throughout the test, at 1.2 mg/L. Therefore, effect parameters were based on initially measured concentrations. The EC50 and EC10 for growth rate inhibition (72h-ErC50 and 72h-ErC10) were >1.2 mg/L being considered the maximum soluble amount of the test item in test medium at a loading rate of 100 mg/L. The 72h-NOErC was 1.2 and <1.2 mg/L based on biological relevance and statistical significance, respectively.

The study met all validity criteria, and is considered reliable without restriction.