Pyrrolidinium, 1-[[(6R,7R)-7-amino-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-1-methyl-, chloride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples (5 mL per replicate) were taken from each test concentration and from the control group at the start and at the end of the test. From the control two replicates, from the treatment groups three replicate samples were taken.

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion. A stock solution was first prepared by dissolving an amount of 0.08 g test item in 800 mL dilution water (OECD medium) resulting a nominal concentration of 100 mg/L. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared just before introduction of algae (start of the experiment).

Test organisms (species):

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

Details on test organisms:

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

According to the guideline.

Post exposure observation period:

No.

Hardness:

Not reported.

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.8 and 23.1°C measured in the flask and in the range of 22.0 – 23.6°C measured within the climate chamber.

pH:

The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 8.23 – 8.83 during the experiment.

Dissolved oxygen:

Not reported.

Salinity:

No.

Conductivity:

Not reported.

Nominal and measured concentrations:

0, 6.25, 12.5, 25, 50 and 100 mg/L nominal.

Details on test conditions:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 8014 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.

The test was performed with three replicates per test concentration and six replicates in the control group.
The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test item solution. The initial cell density was about 104 cells/mL, in each test flask.
Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks. The test flasks were covered with air-permeable stoppers.
The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.

Mean values and standard deviations were calculated for each concentration at each observation period using Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test (α = 0.05; 2-Tailed) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test.
For determination of the ECx values no statistical analysis was necessary (as no significant toxicity was observed).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

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

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

Details on results:

Based on the results of the statistical evaluation neither the average specific growth rate nor the yield showed significant difference compared to the control in any of the treated concentrations.
Accordingly, the 72-h LOEC and EC50 values based on average specific growth rate and yield were determined to be > 100 mg/L.

Results with reference substance (positive control):

The date of the last study (Study number: 392-201-1921) with the reference item Potassium dichromate was: 20 – 23 September 2016.
The 72h ErC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.77 mg/L)
The 72h EyC50: 0.46 mg/L, (95 % confidence limits: 0.42 – 0.49 mg/L)

The nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of MPI-ACA varied between 91 and 115 % of the nominal at the start and between 98 and 127 % at the end of the experiment. In the untreated control group the test item was not detected. The analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 72 hours in most cases. The measured values were slightly above 120 % of the nominal (125 and 127 %) at the end of the test in the two lowest concentrations (6.25 and 12.5 mg/L). However, no toxicity was observed in the higher concentrations (up to 100 mg/L), therefore the biological results can be based on the nominal concentrations.

Validity of the Test

 The cell density in the control cultures increased by a factor of 40.17 within 72 hours. This corresponds to a specific growth rate of 1.23 day-1.

 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: 12.68 %

- CV for section-by-section growth rate day 1-2: 6.93 %

- CV for section-by-section growth rate day 2-3: 10.36 %

The mean coefficient of variation for section-by-section specific growth rates: 9.99 %.

 The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 4.33 %.

All validity criteria were met, therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had no toxic effect up to at least at 100 mg/L concentration (i.e. limit test concentration) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC are higher 100 mg/L.

Executive summary:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had no toxic effect up to at least at 100 mg/L concentration (i.e. limit test concentration) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC are higher 100 mg/L.

Description of key information

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had no toxic effect up to at least at 100 mg/L concentration (i.e. limit test concentration) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC are higher 100 mg/L.

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 EC50 and EC10 are not 100 mg/L, as one is forced to enter in the field above, but the EC50 and EC10 are >100 mg/kg.