Carbazamidine sulphate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Guanidinium phosphate (1:1)

EC Number:

226-551-9

EC Name:

Guanidinium phosphate (1:1)

Cas Number:

5423-22-3

Molecular formula:

CH5N3.H3O4P

IUPAC Name:

Guanidium phosphate (1:1)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations (final test): Blank control, 10, 22, 46, 100 and 220 mg/L
- Sampling
Frequency: at t=0 h and t=72 h
Volume: 1 mL
- Sample storage conditions before analysis: Samples were transported on dry ice to TNO Triskelion (P.O. Box 844, 3700 AV, Zeist, The Netherlands) and were stored in a freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a nominal concentration of 100 mg/L for the combined limit/range-finding test and 220 mg/L for the final test applying a short period of vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the respective nominal concentration in test medium. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
- Controls: Blank control containing test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Microalgae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days (pre-culture)
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1, according to the “Nederlandse Praktijk Richtlijn no. 6505”) 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 21-24°C until the pre-culture started.

PRE-CULTURE:
- Period: 3 days before the start of the test
- Culturing media and conditions (same as test or not): Yes (M2)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

Final test
Start: 22 °C
During test: 21.7 - 23 °C

pH:

Final test
Start: 6.5 - 8.2
End: 6.9 - 8.2

Nominal and measured concentrations:

- Nominal
Final test: Blank control, 10, 22, 46, 100 and 220 mg/L
- Measured
t=0h: <0.08 mg/L* (blank control), 10.1 mg/L (10 mg/L), 24.9 mg/L (22 mg/L), 45.3 mg/L (46 mg/L), 94.6 mg/L (100 mg/L) and 205 mg/L (220 mg/L)
t=48h: <0.08 mg/L* (blank control), 10.2 mg/L (10 mg/L), 22.6 mg/L (22 mg/L), 47.5 mg/L (46 mg/L), 97.6 mg/L (100 mg/L) and 212 mg/L (220 mg/L)
*) LOD was 0.08 mg/L (lowest calibration point).

Details on test conditions:

TEST SYSTEM (final test)
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: None
- Initial cells density: 1 x10^4 cells/mL
- Control end cells density (range): 162.22x10^4 - 216.82x10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (M2, according to OECD 201 guideline)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: No (3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium 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)
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature of medium was measured continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 88 to 89 µE.m-2.s-1. TLD-Iamps of the type 'Cool-white' of 30 Watt

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm. Algal medium was used as blank and the extra replicates as background for the treated solutions. At the end of the test microscopic observations were performed on the 100 mg/L concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations (final test): approximately x2
- Combined limit/range-finding test
- Test concentrations: Blank control, 0.10, 1, 10 and 100 mg/L
- Results of the range finding study used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 57 - 200 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 8.7 - 31 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

other: guanidine content (32.7% w/w)

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 19 - 65 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: guanidine content (32.7% w/w)

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3 - 10 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: guanidine content (32.7% w/w)

Basis for effect:

growth rate

Details on results:

- Final test
Measured concentrations: actual concentrations were in agreement with nominal and remained stable during the test period (93-113%). Given this result the effect parameters were based on the nominal test concentrations.

Biological results: growth rate was in the range of the control at 10 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 22 mg/L and higher were increasingly reduced. Statistically significant reduction of growth rate was found at test concentrations of 22 mg/L and higher. Inhibition of yield increased with increasing concentration of Guanidine phosphate (1:1) from 10 mg/L upwards resulting in >90% inhibition at and above 100 mg/L. Statistically significant inhibition of yield was found at test concentrations of 22 mg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Acceptability of the test:
1. In the control, cell density increased by an average factor of >16 within two days.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Results with reference substance (positive control):

Potassium dichromate: The EC50 for growth rate reduction (ErC50: 0-72h) was 1.1 mg/L (95% confidence interval: 0.71 to 1.6 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Guanidine phosphate (1:1) reduced growth rate of this fresh water algae species significantly at 22 mg/L and higher. The EC50 for growth rate reduction (72h-ErC50) was 110 mg/L. The 72h-NOEC for growth rate reduction was 10 mg/L.

Executive summary:

In a 72-h toxicity study conducted according to OECD guideline 201, microalgae (Pseudokirchneriella subcapitata) were exposed to Guanidine phosphate (1:1) under static conditions at the following nominal concentrations (final test): 0 (blank control), 10, 22, 46, 100 and 220 mg/L. The test substance significantly reduced growth rate at 22 mg/L and higher. The 72-h EC50 for growth rate reduction was 110 mg/L (corresponding to 36 mg guanidine/L) and the 72h-NOEC for growth rate reduction was 10 mg/L (corresponding to 3.3 mg guanidine/L) based on analytically confirmed nominal concentrations. The study is considered to be reliable without restrictions.