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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:
30 Apr 2018 - 09 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
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: all test concentrations and the control
- 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 100 mg/L (corrected for water content) 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
- The batch of test item tested was a clear and almost colourless liquid and completely soluble in test medium at the concentrations tested. A correction was made for the water content of the test item. All reported concentrations are based on the pure test item.
- Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15-minute period of magnetic stirring to accelerate dissolution of the test item in medium. After adjusting the pH from 10 to 8.4, the solution was used as the highest test concentration. 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: 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 stock culture 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 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.
- Pre-culture medium and test medium: M2 medium, according to OECD 201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21 - 23°C
pH:
Control: 8.2 (t=0 h); 8.4 (t=72 h)
100 mg/L: 8.3 (t=0 h); 8.3 (t=72 h)
Nominal and measured concentrations:
Nominal: 0.10, 1.0, 10 and 100 mg/L and control.
Only samples from the highest test concentration and the control were analysed. The measured concentrations were at 102-109% of the nominal concentrations throughout the test, i.e. were between 102 and 109 mg/L at the start and at the end of the test in the samples with and without algae. Based on these results, the nominal exposure concentrations were used to determine the effect parameters.
Details on test conditions:
The test was performed as a Combined Limit/range-finding Test.

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: 270 x 10^4 cells/mL
- Replicates: 6 replicates of the control and the highest test concentration, 3 replicates of each intermediate test concentration, 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 the limit concentration and the control. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not during test (only during preparation of test solutions).
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 79 - 83 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. 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.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the 100 mg/L and the control to observe for any abnormal appearance of the algae.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Mar 2018)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
growth rate
Details on results:
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 100 mg/L when compared to the control.
- Growth rates were in the range of the control at the three lowest test concentrations, whereas a statistically significant inhibition of growth rate (3.2%) exposed to the highest test concentration was observed. Since the effect was below 10%, and thus considered to be biologically not relevant, the NOEC was set at 100 mg/L.
- Analytical results: A small response was measured in the control at the end of the test. The source of this response could not be identified. Considering that the validity criteria were met and that the concentration could only be estimated by extrapolation of the calibration curve (i.e., fell outside the calibration curve), this was considered to be negligible. The maximum contribution to the lowest sample concentration was 0.05%, taking the dilution factor into account.
- 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 1.6 mg/L with a 95% confidence interval ranging from 1.5 - 1.6 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:
NOEC and ECx calculations:
An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate (STUDENT-t test for Homogeneous Variances, α=0.05, one-sided, smaller). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Combined Limit/Range-finding Test: Test Samples

Time of sampling
[hours]

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

n.d.

n.a.

 

 

100

104

104

 

 

 100(a)

107

107

 

24

0

n.d.

n.a.

n.a.

 

100

106

106

102

 

 100(a)

108

108

101

72

0

0.061(b)

n.a.

n.a.

 

100

109

109

105

 

 100(a)

102

102

95

a     Without algae.

b     Estimated value, calculated by extrapolation of the calibration curve. The maximum contribution to the lowest sample concentration was 0.05%, taking the dilution factor into account.

n.d. Not detected.

n.a. Not applicable.

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


Test item Nominal conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.866

0.0117

6

 

0.10

1.863

0.0025

3

0.14#

1.00

1.870

0.0031

3

-0.22#

10.00

1.863

0.0050

3

0.11#

100.00

1.805

0.0157

6

3.2*

* Effect was statistically significant but considered to be biologically not relevant (<10%).
#Not statistically compared to the control.

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

Test item Nominal conc. (mg t.i./L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.431

 

1.808

 

1.358

 

100

6

2.509

-3.2

1.717

5.0

1.19

12

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 72h-ErC50 and 72h-ErC10 were beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L (corrected for water content).
The 72h-NOEC for growth rate inhibition was 100 mg/L (corrected for water content).
Executive summary:

In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 100 mg/L and an untreated control (both 6 replicates). Intermediate concentrations of 0.10, 1.0 and 10 mg/L were tested in triplicate. A correction was made for the water content of the substance, i.e. all reported concentrations are based on the pure test substance.

Measured concentrations of the substance were stable at 102 -109% of nominal throughout the test. Nominal exposure concentrations were used to determine the effect parameters. No biologically relevant inhibition of growth rate was recorded at any of the concentrations tested. The 72h-ErC50 and 72h-ErC10 were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition was 100 mg/L. The study met all validity criteria, and is considered reliable without restriction.

Description of key information

No biologically relevant inhibition of growth rate was recorded at any of the concentrations tested. The 72h-ErC50 and 72h-ErC10 were beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L (corrected for water content).

The 72h-NOEC for growth rate inhibition was 100 mg/L (corrected for water content).

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 100 mg/L and an untreated control (both 6 replicates). Intermediate concentrations of 0.10, 1.0 and 10 mg/L were tested in triplicate. A correction was made for the water content of the substance, i.e. all reported concentrations are based on the pure test substance.

Measured concentrations of the substance were stable at 102 -109% of nominal throughout the test. Nominal exposure concentrations were used to determine the effect parameters. No biologically relevant inhibition of growth rate was recorded at any of the concentrations tested. The 72h-ErC50 and 72h-ErC10 were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition was 100 mg/L. The study met all validity criteria, and is considered reliable without restriction.