Ammonium benzoate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2018 - 11 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium chloride

EC Number:

235-186-4

EC Name:

Ammonium chloride

Cas Number:

12125-02-9

Molecular formula:

ClH4N

IUPAC Name:

ammonium chloride

Test material form:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
608031
- Expiration date of the lot/batch:
25. Oct. 2018
- Purity test date:
25. Oct. 2016 (CoA)

RADIOLABELLING INFORMATION (if applicable)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
closed vessel dark and dry at room temperature 20 ± 5°C
- Stability under test conditions:
assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
assumed soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
assumed non-reactiv

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
no specific treatment

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Because of insufficient stability of the test item in test medium, analytical measurement was performed daily. Stability in the test solutions with algae after 3 days was lower than in the solutions without algae. That means test item was present in the test solution, but partly not measurable in presence of algae cells because of ingestion or adsorption of test item onto the algae cells. Therefore, the analytic determination in this study is performed only in test solutions without algae.

No sample preparation was performed. Only the control solutions were filtrated over 0.45 µm PTFE membrane syringe filters before analytical measurement.

On each sampling day, validity of calibration was controlled by measuring QC samples. The recovery of the QC samples was in the stated range.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution containing 100.4 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. Lower test concentrations were prepared by dilution of the stock solution in algal medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

6.3.1 Specification
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
6.3.2 Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

not stated

Test temperature:

22.0 – 22.8 °C

pH:

7.6 - 8.3

Dissolved oxygen:

not stated

Salinity:

not stated

Conductivity:

not stated

Nominal and measured concentrations:

The measured concentrations lay between 106 % and 100 % of the nominal concentra-tions at the beginning of the test and between 90 % and 71 % of the nominal concentration after the first 24 hours. No test item could be detected after 48 h and 72 h. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The measured concentrations after 0 h and 24 h and the LOD of 2 mg/L for the measuring times 48 and 72 h were used for this purpose.
Geometric mean is calculated by multiplication of the n participating concentrations and taking the nth root.
The details are given in tables in section "any other information on materials and methods incl. tables"

Details on test conditions:

Date: 23. – 26. Apr. 2018
Treatments tested: 4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration
The concentrations to be tested based on non GLP pre- tests
Replicates
Blank Control: 6 replicates, filled with 45 ±1 mL algal medium and algae
Treatments: 3 replicates, filled with 45 ±1 mL test solution and algae
1 replicate, filled with 45 ±1 mL test solution without algae
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: 5000 lux
Temperature: 22.0 – 22.8 °C

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

no abnormal cell shapes noted.

Validity
Parameter Validity criteria Observed value Assessment
Increase factor biomass factor 16 in 72 h 107 Valid
Mean coefficient of variation of max. 35% 16% Valid
daily growth rates
Coefficient of variation of average max. 7% 1% Valid
growth rate during the whole test period

Results with reference substance (positive control):

The results of the last study with the positive control K2Cr2O7 are presented in the following table. The study was performed under GLP conditions in August and September 2017.
Biological Results of K2Cr2O7
Parameter Value 95% confidence interval
72h ErC50 0.90 mg/L 0.77 – 1.1 mg/L
72h EyC50 0.42 mg/L 0.35 - 0.51 mg/L

Reported statistics and error estimates:

Parameter Value 95% confidence interval
72h ErC10 13.67 mg/L 10.71 – 17.44 mg/L
72h EyC10 4.59 mg/L 2.51 – 8.37 mg/L
72h ErC50 > 13.10 --
72h EyC50 18.54 mg/L 7.16 – 45.44 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, Ammonium Benzoate showed effects on the growth of Algae
with an EC50 (Growth Rate) > 13.10 mg/L and EC50 (Yield) = 18.54 mg/L.

Executive summary:

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L. Incubation time (test systemDesmodesmus subspicatus)was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1]were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following nominal concentrations: 10 mg/L - 100 mg/L.

 

Because of the insufficient stability of the test item in algal medium, the content of the test item in the test solutions was determined daily using HPLC. According to the results of the validation (see chapter6.6) the stability of the test item in the test solution with algae after 3 days was lower than in the solutions without algae. That means test item was present in the test solution, but not measurable in presence of algae cells because of ingestion or adsorption of test item onto the algae cells. Therefore, one additional replicate per concentration with test solution and without algae was prepared for the analytical determination throughout the test.

The measured concentrations lay between 106% and 100 % of the nominal concentrations at the beginning of the test and between 90% and 71 % of the nominal concentration after the first 24 hours. No test item could be detected after 48 h and 72 h. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The measured concentrations after 0 h and 24 h and the LOD of 2 mg/L for the measuring times 48 and 72 h were used for this purpose.

According to the statistics program, no EC₅₀value can be determined for growth rate. Therefore, this value is specified as a range.

 

The 72h-EC50s of potassium dichromate (K₂Cr₂O₇, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

[1]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 8.2 Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.

The following results for the test item Ammonium Benzoatewere determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	3.00 mg/L	4.28 mg/L	13.67 mg/L	>13.10 mg/L
Yield	3.00 mg/L	4.28 mg/L	4.59 mg/L	18.54 mg/L