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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:
25 - 29 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
5 March 2015
Specific details on test material used for the study:
- Relative Density: 1.196 (pycnometer method, OECD Guideline 109, EU Method A.3)
Analytical monitoring:
yes
Details on sampling:
Concentration of dissolved organic material was checked by analysis of the Chemical Oxygen Demand (ST-COD). Regarding properties of the test item, COD analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.
Duplicate abiotic samples (since no significant differences were observed between chemical analyses in abiotic and biotic samples in the range-finding test, and to avoid COD increase due to algal development) for analysis were taken from the control and the loading rate of 100 mg/L at the start and every day thereafter until the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at 50 °C, followed by sonication for 5 minutes. Based on experience on similar substances, these steps (heating and sonication) were essential to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5 x 10^3 cells/mL per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions (WAFs).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Stock culture: Algae stock cultures were started by inoculating growth medium (=test water) 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 23 ± 2 °C.
- Pre-culture 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
23.1-24.1 °C throughout the test (average value: 23.2 °C)
pH:
8.10 (0 h); 9.29 (72 h)
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentration: 100 mg/L (loading rate)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type: Closed
- Initial cells density: An initial cell density of 5 x10^3 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 6 replicates
- No. of vessels per control (replicates): 6 replicates
* Moreover, replicates of each treatment without algae will be prepared for chemical analyses.

TEST MEDIUM / WATER PARAMETERS
- Test water: Original medium of OECD TG 201; Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity was 4761 lux (range: 4675-4832 lux)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0 h and t=72 h).
Temperature of Medium: Measured continuously in the growth chamber, over the study period.
Light Intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Range finding study: A range-finding test was conducted to determine the range of concentrations for the definitive test. Algal cells were exposed to the nominal concentrations (in triplicate) of 1.0, 10, 100 and 1000 mg/L (loading) and to a control for 72 hours. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.
- Results used to determine the conditions for the definitive study: Algal growth inhibition at 72 hours was 0.2, 1.0, 1.2 and 8.7% at 1.0, 10, 100 and 1000 mg/L, respectively. Based on the results of a range-finding test, a limit test was performed at 100 mg/L (loading).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- After 24, 48 and 72 hours of exposure an inhibition of algal growth relative to control values was observed at 100 mg/L (loading). Nevertheless, this was a slight inhibition with 5% growth inhibition relative to the control at t=72 h.
- However, it should be noted that previous non-GLP tests showed a stronger inhibition of algal growth at a loading rate of 100 mg/L. The applied pre-treatment, and more particularly the duration of sonication, was suspected to be the cause of this growth inhibition. Indeed, when sonication was carried out for about 10 min, the colour of the water column observed after preparation (heating / sonication / slow-stirring during 24 h) of the loading rate was slightly yellow in comparison with the control or a preparation with a shorter time of sonication. These observations were confirmed in the present study by the preparation of another loading rate of 100 mg/L sonicated for 10 min (instead of 5 min as the other one), where 43% growth inhibition relative to the control was observed at t=72 h. Thus, a physical effect related to the duration of the physical treatment was suspected to be the source of growth inhibition rather than toxicity due to the test substance. Nevertheless, regarding results of COD content in WAFs (ca. 50 mg O2/L) an effect directly due to a test item effect is not excluded. In all cases, based on these results, the estimated 72-hour EL50 should be therefore > 100 mg/L (loading).
Results with reference substance (positive control):
On January 29, 2016 (most recent test), the 72 h-EC50 was 0.84 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72 h-EC50: 0.60 to 1.03 mg/L).
Reported statistics and error estimates:
The software ToxRat® Professional was used to perform statistical analyses. Complementary statistical analyses were performed using another statistical spreadsheet (using Excel®) for the validity criteria of the study.

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mLx104)

Time

Replicate

Nominal concentration(mg/L)*

Control

100

t=24 h

1

7.2

4.0

2

8.0

4.8

3

5.6

5.2

4

5.2

5.6

5

5.2

4.8

6

4.4

4.4

Mean

5.9

4.8

Std. Dev.

1.37

0.57

% Inhibition

-

8.0

t=48 h

1

17.6

12.0

2

17.2

14.0

3

16.8

12.8

4

20.4

12.0

5

14.0

12.4

6

17.6

11.6

Mean

17.3

12.5

Std. Dev.

2.05

0.85

% Inhibition

-

9.1

t=72 h

1

66.4

51.2

2

63.6

48.8

3

62.8

49.6

4

63.2

50.8

5

65.2

51.2

6

62.8

51.6

Mean

64.0

50.5

Std. Dev.

1.48

1.09

% Inhibition

-

4.9

* WAF prepared at the given loading rate.

%Inhibition of growth rate relative to the control determined by the computer program ToxRat.

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 6 replicates of each test concentration.

Std. Dev.: standard deviation

Analytical results:

Concentration of dissolved organic material in the control and the loading rate of 100 mg/L was checked by analysis of the Chemical Oxygen Demand (ST-COD) at the start and every day thereafter until the end of the test.

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 14 mg O2/L in the WAFs at 100 mg/L. Nevertheless, these results show that WAFs concentrations were overall stable between the start and the end of the test. Since the study was carried out using WAFs of a natural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing, the results were based on the nominal WAFs concentrations (72-hour ELx values for the parameters growth rate and yield, Effect Loading).

Table 6.1.5/2: Concentrations of the test substance in test water - results of the determination of the COD analysis in the final test

 

COD (mg O2/L)

Nominal concentration* (mg test item/L)

Start (t=0h)

t=24h

t=48h

End (t=72h)

 

Mean

 

Mean

 

Mean

 

Mean

Control

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

Control

<LOQ

<LOQ

<LOQ

<LOQ

100

14

14

13

13

14

14

12

13

100

14

13

14

13

 * WAF prepared at the given loading rate

 

Physico-chemical parameter values throughout the test:

The temperature in the incubator was situated between 23.1 and 24.1 °C throughout the test (average value: 23.2 °C). The mean light intensity was of 4761 lux (range: 4675-4832 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23 ± 2 °C, constant within 2 °C; light intensity: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

 

Validity criteria of the study:

Cell density in Controls: 128-fold increase within 72 hours

Coefficient of Variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 14%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 0.5%.

Thus the validity criteria were respected in this study.

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg/L (loading). The 72-hour NOEC was estimated to be higher than 100 mg/L (loading).
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, algal cells (Pseudokirchneriella subcapitata) were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start and every day thereafter until the end of the test. Definitive test was performed based on the results of range-finding test. 

 

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 14 mg O2/L in the WAFs at 100 mg/L and that these levels were stable throughout the exposure. After 24 and 48 hours of exposure a very slight inhibition of algal growth relative to control values (approximately 10%) was observed at 100 mg/L (loading), confirming the observations of the range-finding test where results showed few or no effect at the test loading rates 1 to 100 mg/L.

 

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg/L (loading). The 72-hour NOEC was estimated to be higher than 100 mg/L (loading).

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

WAF, 72h-ErL50 (Pseudokirchneriella subcapitata) > 100 mg/L (loading)

WAF, 72h-NOEC (Pseudokirchneriella subcapitata) > 100 mg/L (loading)

Key value for chemical safety assessment

Additional information

One key study is available to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to the OECD Guideline 201 with GLP statement. Following a preliminary range-finding test, the algae were exposed to Water Accommodated Fractions (WAFs) of the test substance at a nominal loading rate of 100 mg/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start and every day thereafter until the end of the test. Although every effort was made in a first time to extract and solubilise the soluble fraction of the test substance in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 14 mg O2/L in the WAFs at 100 mg/L (although this is not unusual as non-soluble fractions are excluded from WAFs) and that these levels were stable throughout the exposure. After 24, 48 and 72 hours of exposure a very slight inhibition of algal growth relative to control values (<10%) was observed at 100 mg/L (loading), confirming the observations of the range-finding test where results showed few or no effect at the test loading rates 1 to 100 mg/L. Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg/L (loading). The 72-hour NOEC was estimated to be higher than 100 mg/L (loading).