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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:
14 Aug 2017 - 21 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006, Annex 5 corrected 2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control, at t=0 h, t=24 h and t=72 h. Only samples at the limit concentration were analysed.
- Sampling method: 2.0 mL. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at highest concentration 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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying a 20-minute period of magnetic stirring followed by a 18-minute period of ultrasonic waves 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 (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 1E+04 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
- Pre-culture medium and test medium: M2, according to OECD 201.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22 - 23°C.
pH:
8.0-8.1
Nominal and measured concentrations:
Nominal: 0.10, 1.0, 10 and 100 mg/L
Measured (only nominal concentration of 100 mg/L, with algae): 112 mg/L (t=0 h), 111 mg/L (t=24 h), 111 mg/L (t=72 h).
Measured (only nominal concentration of 100 mg/L, without algae): 113 mg/L (t=0 h), 113 mg/L (t=24 h), 103 mg/L (t=72 h).

Measured concentrations were 103-113% of nominal, therefore effect parameters were expressed in terms of analytically confirmed nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: capped 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1E+4 cells/mL
- Control end cells density: 280.5E+4 cells/mL
- Replicates:
* 6 replicates of the control and the highest concentration.
* 3 replicates of the lower concentrations.
* 1 or 2 replicates of each test concentration without algae.
* 1 or 2 replicates of each test concentration for sampling purposes at 24 hours.
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes, M2 according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 in the control and the limit concentration. Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 80 to 84 μE.m-2.s-1.

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 (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Sep 2017)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
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.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Based on statistical significance
Details on results:
- No biologically relevant inhibition of growth rate was recorded at any of the concentrations tested.
- 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 highest concentration when compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 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:
STUDENT-t test for Homogeneous Variances, α=0.05, one-sided, smaller. Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Test item

Nominal concentration (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.878

0.0303

6

 

0.10

1.855

0.0203

3

1.2

1.0

1.812

0.0314

3

3.5

10

1.845

0.0099

3

1.7

100

1.763

0.0247

3

6.1#

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


Table 2: Measured concentrations in test samples

Time of sampling
[hours]

Date of sampling

Date of
analysis (a)

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

14 Aug 2017

21 Aug 2017

0

n.d.

n.a.

 

 

 

 

100

112

112

 

 

 

 

 100 (b)

113

113

 

24

15 Aug 2017

21 Aug 2017

0

n.d.

n.a.

n.a.

 

 

 

100

111

111

99

 

 

 

 100 (b)

113

113

99

72

17 Aug 2017

21 Aug 2017

0

0.010 (c)

n.a.

n.a.

 

 

 

100

111

111

99

 

 

 

 100 (b)

103

103

91

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

(b)   Without algae.

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

n.d. Not detected.

n.a. Not applicable.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The EC50 for growth rate inhibition (72h-ERC50) was >100 mg/L. The 72h-NOErC was 100 mg/L (based on biological relevance).
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 0.1, 1.0, 10 and 100 mg/L. Measured concentrations were at the level of nominal (103 -113%), therefore the effect parameters were based on nominal concentrations. No biologically relevant inhibition of growth rate was recorded at any of the concentrations. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L. The 72h-NOErC was 100 mg/L based on biological relevance. The study met all validity criteria and is considered reliable without restriction.

Description of key information

In a study according to OECD 201, the 72h-EC50 for growth rate inhibition was determined to be >100 mg/L, based on analytically confirmed nominal concentrations. The 72h-NOErC was 100 mg/L based on biological relevance (as an inhibition of <10% (6.1%) was observed at 100 mg/L).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

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 0.1, 1.0, 10 and 100 mg/L. Measured concentrations were at the level of nominal (103 -113%), therefore the effect parameters were based on nominal concentrations. No biologically relevant inhibition of growth rate was recorded at any of the concentrations. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L. The 72h-NOErC was 100 mg/L based on biological relevance, as an inhibition of <10% (6.1%) was observed at 100 mg/L. The study met all validity criteria and is considered reliable without restriction.