1-(4-cyanophenyl)guanidine

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:

2015-09-28 to 2015-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to standard test guidelines (OECD, EC) and conform GLP requirements. The light intensity was not recorded but since all the validity criteria were met, it can be concluded that the study integrity was not adversely affected by this deviation. The results of the study can be considered reliable without restriction.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(4-cyanophenyl)guanidine

EC Number:

611-382-9

Cas Number:

5637-42-3

Molecular formula:

C8H8N4

IUPAC Name:

1-(4-cyanophenyl)guanidine

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as stated in study reports): JNJ-4508530-AAA (T002707)
- Physical state: solid (powder)
- Appearance: white, beige powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WIL Research Europe, I15BB0573
- Expiration date of the lot/batch: 08 February 2017 (retest date)
- Appearance: White, beige powder (determined by WIL Research Europe at receipt of the test item)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test and after 72 hours from all test concentrations and from the control. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal test item concentration of 100 mg/L was prepared applying 10 to 15 minutes of magnetic stirring followed by a 5 to 7 minute treatment period with ultrasonic waves. Thereafter the pH was adjusted to 8.3 with 1M HCl. Adequate volumes of the highest test concentration were diluted with test medium to prepare the test media with the lower test item concentrations.
- Controls: yes

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal stock cultures were started by inoculating growth medium 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

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:

21-24°C

pH:

7.7 - 8.2

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

nominal test concentrations final test 1: 10, 18, 32, 56 and 100 mg/L
measured test concentration final test 1 t= 0 h: 8.60, n.d, n.d, n.d and 101 mg/L
measured test concentration final test 1 t= 72h: 10.9, n.d, n.d, n.d and 107 mg/L

nominal test concentrations final test 2: 1.0, 3.2, 10, 32 and 100 mg/L
measured test concentration final test 2 t= 0 h: 0.941, 3.00, 9.35, 28.1 and 95.2 mg/L
measured test concentration final test 2 t= 72h: 0.952, 3.06, 9.48, 30.9, 91.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 187.4 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: the pH of the 100 mg/L concentration was adjusted from to 8.3 with 1M HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 µE.m-2.s-1 in the photosynthetically effective wavelength range of 400 to 70 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
* final test 1: 1.8
* final test 2: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0 and 10 mg/L
- Results 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.:

83 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 80-87 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: clear test solution
- EC50 (yield) = 22 mg T002707 /L (95% C.L. 19-24 mg/L)
- EC10 (growth rate) = 15 mg T002707 / L (95% C.L. 13-16 mg/L).
- EC10 (yield) = 11 mg T002707 / L (95% C.L. 7.9-13 mg/L).
- NOEC (yield) = 10 mg T002707 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L (95% CI 1.1 to 1.2 mg/L)

Reported statistics and error estimates:

NOEC and EC values were determined using a Williams Multiple Sequential t-test Procedure (alpha=0.05, one-sided, smaller).
Calculation of ECx values was based on probit analysis using linear max. likelihood regression.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T002707 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T002707 inhibited growth rate of this fresh water algae species significantly at 32 mg/L and higher. The 72-h ECr50 was 83 mg/L (95% C.L. 80-87 mg/L).
The results of the test can be considered reliable without restrictions.