2,2'-(piperazine-1,4-diyl)bis(ethanesulphonic) acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-25 to 2017-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

March 01, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: For determination of the test item concentration, five replicate samples were taken from the test item treatment group and two from the control at the start and six replicate samples were taken from the test item treatment group and two from the control at the end of the experiment. The analysis was performed using HPLC method with UV detection.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution used in the test was prepared by mechanical dispersion. An amount of 0.08 g test item was dissolved in 800 mL dilution water (OECD medium) in order to obtain the concentration of 100 mg/L and this solution was placed into the ultrasonic bath for 5 minutes. The test solution was freshly prepared in the testing laboratory just before introduction of algae (start of the experiment).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source: SAG: Collection of Algal Cultures, 37073 Göttingen, Germany
- Method of cultivation: algae were kept as stock culture on solid agar

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

22.3 - 22.4 °C (measured in the flasks) and
22.1 - 23.8 °C (in the climate chamber)

pH:

7.85 – 8.64 (in the control group) and 6.54 - 6.95 (in the treatment group during the experiment)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal conc.: 100 mg/L
Measured conc.: The measured concentrations showed very good correlation with the nominal concentrations. The recovery rate in the five treated groups after 72 hours was 101 % - 102 %.
Therefore the nominal concentration was used for the determination of the results.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks 250 mL
- Type: air-permeable stoppers
- Material, size, fill volume: glass, 205 mL, 100 mL fill volume
- Initial cells density: 10000 cells/mL
- Control end cells density: approx. 4633000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (acc. to OECD TG 201)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7117 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: manual cell counting using a microscopic method with a counting chamber at 24, 48 and 72 h

TEST CONCENTRATIONS
- Range finding study: yes (6 concentrations ranging from 0.001 to 100 mg/L)
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: none reported
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? YES
- EC50: The 72 h ErC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.77 mg/L)
The 72 h EyC50: 0.46 mg/L, (95 % confidence limits: 0.42 – 0.49 mg/L)

Validity of the Test

- The cell density in the control cultures increased by a factor of 46.33 within 72 hours. This corresponds to a specific growth rate of 1.28 /day.

-The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 25.28 %

- CV for section-by-section growth rate day 1-2: 18.76 %

- CV for section-by-section growth rate day 2-3: 9.18 %

The mean coefficient of variation for section-by-section specific growth rates: 17.74 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 3.41 %.

All validity criteria were met. Therefore, the study was considered as valid.

Analytical Results

The measured concentration of the test item was 99 % of the nominal at the start and 101 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 72 hours. Therefore the biological results were based on the nominal concentration.

Biological Results

Slight inhibition was observed in the treated group (2.2 % inhibition for growth rates and 8.8 % for yield) after 72 hours of exposure compared to the control which was considered to be within the normal biological range.

Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test (α = 0.05, SPSS software). The results of the statistical evaluation showed that the 0 – 72 h average specific growth rate and yield at the treatment group (100 mg/L) were not statistically significantly different from the untreated control. Because the test item had no significant effect on the growth of algae, no statistical analysis was performed to determine EC values.

Summary of the Biological Endpoints

Parameter (0 - 72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
EC50 95 % conf. limits	> 100 not calculated	> 100 not calculated
NOEC	100	100
LOEC	> 100	> 100

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, no effects were observed on the algal cells. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100 mg/L. The 72 h EC50 values based on growth rate and yield were determined to be > 100 mg/L.

Executive summary:

The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a study according to OECD 201 under GLP conditions. The alga was exposed to the test item for 72 hours. Based on the results of a non-GLP preliminary test, in which the test item had no toxic effect on algae up to the limit concentration of 100 mg/L, the main test was performed with 100 mg/L as a limit test. In parallel with the test item treatments, potassium dichromate was used as reference control. As measured test item concentration remained within ± 20 % of the nominal concentration over the test period of 72 hours, the biological results were based on the nominal concentration. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations in a static test. The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. Per test concentration and control, six replicates were used. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test. The results of the statistical evaluation showed that the 0 – 72 h average specific growth rate and yield in the treatment group were not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100 mg/L. Consequently, the 72 h EC₅₀ values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.

Description of key information

Under the test conditions, no effects were observed on the algal cells. The NOEC was determined to be 100 mg/L and the 72 h EC₅₀ values based on growth rate and yield were determined to be > 100 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a study according to OECD 201 under GLP conditions. The alga was exposed to the test item for 72 hours. Based on the results of a non-GLP preliminary test, in which the test item had no toxic effect on algae up to the limit concentration of 100 mg/L, the main test was performed with 100 mg/L as a limit test. In parallel with the test item treatments, potassium dichromate was used as reference control. As measured test item concentration remained within ± 20 % of the nominal concentration over the test period of 72 hours, the biological results were based on the nominal concentration. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations in a static test. The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. Per test concentration and control, six replicates were used. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Student’s t-test. The results of the statistical evaluation showed that the 0 – 72 h average specific growth rate and yield in the treatment group were not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100 mg/L. Consequently, the 72 h EC₅₀ values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.