N,N'-diphenylguanidine monohydrochloride

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:

from 2018-08-09 to 2018-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations three replicate samples (3 mL per replicate) were taken from each test concentration and six replicates (3 mL per replicate) from the control group at the start and at the end of the test.

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 0.025 g test item in 500 mL dilution water (OECD Medium) resulting a nominal concentration of 50 mg/L. Test solutions were prepared by appropriate dilution of this stock solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

in the flasks: 22.9 - 23.3 °C
in the climate chamber: 22.3 - 23.8 °C

pH:

7.24 - 8.41

Nominal and measured concentrations:

Nominal concentrations: 0.55, 1.4, 3.7, 9.6 and 25 mg/L
Measured concentrations: within ± 20 % of the nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: covered with air-permeable stoppers
- Material, size, headspace, fill volume: glass, ~250 mL volume, 100 mL test medium
- Initial cells density: 1E+4 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (1E+7 algal cells per mL)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD Medium prepared according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7991 lux, fluorescent lamps (range 400 - 700 nm)

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: 3.8 % inhibition at 1 mg/L, 69.2 % inhibition at 10 mg/L and 100 % inhibition at 100 mg/L. Therefore, the EC50 lies between 1 and 10 mg/L.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: microscope with counting chamber at 24, 48 and 72 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.4 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.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The obtained results showed that the test item had inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth were determined to be 5.1, 6.4 and 10.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 2.6, 3.5 and 6.0 mg/L, respectively. The NOEC value based on growth rate and yield was determined to be 3.7 mg/L.

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate was: 26 February – 01 March 2018. The following results were obtained:
The 72h ErC50: 1.08 mg/L, (95 % confidence limits: 0.79 – 1.51 mg/L)
The 72h EyC50: 0.61 mg/L, (95 % confidence limits: 0.49 – 0.76 mg/L)

Preliminary test

After 72 hours of exposure a significant effects on the growth of the algae was observed. At concentration level 10 mg/L, 69.2 % inhibition was observed, while at 1 mg/L only 3.8 % of inhibition was observed. The concentrations for the main test were based on these results.

Table 1: Results of the preliminary range-finding test

Nominal concentrations [mg/L]	Untreated control	0.1	1	10	100
Average cell number at 72 hours (x 1E+4 cells/mL)	141.3	179.0	116.0	5.0	1.0
Growth Rates (µ) [0-72h]	0.0686	0.0720	0.0660	0.0211	0.0000
% Inhibition of µ [0-72h]	–	-5.1	3.8	69.2	100.0
Yield (Y) [0-72h]	140.3	178.0	115.0	4.0	0.0
% Inhibition of Y [0-72h]	–	-26.8	18.1	97.1	100.0

 

Main Test

Analytical results - The measured concentrations of the test item varied between 84 and 101 % of the nominal concentrations (i.e. within ± 20 %) during the experiment, therefore, the biological results are based on the nominal concentrations. In the untreated control group the test item was not detected.

Growth inhibition - The results of the statistical evaluation show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentrations of 9.6 and 25 mg/L and not significantly different in the concentrations of 0.55, 1.4 and 3.7 mg/L. Accordingly, the 72-h NOEC based on growth rate was determined to be 3.7 mg/L and the LOEC was determined to be 9.6 mg/L. The 72-h EC10, EC20, EC50 values for growth were determined to be 5.1, 6.4 and 10.0 mg/L, respectively.

Shape of the algal cells - No abnormal shape of algal cells was noticed in the control and in the three lowest concentrations of 0.55, 1.4 and 3.7 mg/L during the test. In the concentration of 9.6 mg/L mostly thin cells were observed together with normal cells during the test, furthermore some swollen cells were also noticed at the 72-h observation period. In the highest concentration of 25 mg/L thin cells were observed during the test.

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 inhibitory effects on the growth of the algae. The 72-h EC10 and EC50 for growth were determined to be 5.1 and 10.0 mg/L, respectively. The NOEC value based on growth rate and yield was determined to be 3.7 mg/L.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1E4 cells/mL for a period of 72 hours. A concentration series of 0.55, 1.4, 3.7, 9.6 and 25 mg/L and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 84 - 101 % of the nominal concentration. The measured concentrations did not deviate more than 20 % from the nominal concentrations, therefore, the biological results were based on the nominal concentrations. The 72-h EC10, EC20, EC50 for growth were determined to be 5.1, 6.4 and 10.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 2.6, 3.5 and 6.0 mg/L, respectively. The NOEC value based on growth rate and yield was determined to be 3.7 mg/L.The test item showed a significant toxic effect on the green unicellular algae.

Description of key information

In a 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had inhibitory effects on the growth of the algae. The 72-h EC10 and EC50 for growth were determined to be 5.1 and 10.0 mg/L, respectively. The NOEC value based on growth rate and yield was determined to be 3.7 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

5.1 mg/L

Additional information

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1E4 cells/mL for a period of 72 hours. A concentration series of 0.55, 1.4, 3.7, 9.6 and 25 mg/L and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 84 - 101 % of the nominal concentration. The measured concentrations did not deviate more than 20 % from the nominal concentrations, therefore, the biological results were based on the nominal concentrations.The 72-h EC10, EC20, EC50 for growth were determined to be 5.1, 6.4 and 10.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 2.6, 3.5 and 6.0 mg/L, respectively. The NOEC value based on growth rate and yield was determined to be 3.7 mg/L.The test item showed a significant toxic effect on the green unicellular algae.