Isobutyl 4-chloro-3,5-diaminobenzoate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-30 - 2016-08-04 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

version dated 13-Apr-2004

Deviations:

yes

Remarks:

One more treatment was used in the main test than indicated in the study plan (6 instead of 5 treatments). This represents a deviation of the study plan which is not considered to have an influence on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid

Details on test material:

- Other: Storage conditions: Ambient temperature, protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5, 10, 20, 40, 80 mg/L
- Sample storage conditions before analysis: The supporting analyses could not be performed on the same day of collection of the specimens. Therefore, these specimens from freshly prepared test solutions were stored deep-frozen, thawed over-night and then analysed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed in ELENDT M4 dilution water whilst stirring on a magnetic stirrer and shaking on a shaking machine at room temperature. Additionally ultrasonic treatment for 4*10 minutes was necessary to dissolve the test item sufficiently in the test medium. Before weighing the substance into the vessels for treatment preparation it was milled in a mortar made of porcelain.
- Controls: medium without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS (clone 5)
- Source: obtained from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique Appliquee, France in 1978
- Feeding during test: no

ACCLIMATION
- Acclimation period: The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days followed by feeding.
- Type and amount of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Health during acclimation (any mortality observed): none stated

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnias used for the test are not older than 24 hr. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 hr.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Test temperature:

20 ± 1°C

pH:

7.89 - 8.33 during definitive main test (8.02 - 8.85 during screening test)

Dissolved oxygen:

8.2 - 8.5 mg O2/L during definitive main test (8.2 - 8.5 during screening test)

Nominal and measured concentrations:

Nominal: 0, 5, 10, 20, 40, 80, 100 mg/L
Found (t0): 0.00, 4.92, 9.38, 18.90, 36.40, 81.28 mg/L
Found (t48h): 0.00, 4.91, 9.52, 17.41, 33.63, 69.33 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: 50mL, glass
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 (test medium according to OECD 202); the stock solutions with the macronutrients were individually prepared in ultrapure water (for each salt a separate solution). From these individual stock solutions, the medium was prepared.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hr of light and 8 hr of darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility, daily

VEHICLE CONTROL PERFORMED: yes, medium only

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/L

Reference substance (positive control):

yes

Remarks:

To verify the sensitivity of the test system the effect of K2Cr2O7 towards different animals of this breed was tested prior to beginning of the test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

8.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

10.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no immobilisation noted
- Other adverse effects control: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Limit test: no
- Dose-response test: yes
- ECx: EC50 (24 hr) = 0.9 mg/L
- Other: In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test. The value of the EC50 (24 hr) in this period of time was at 0.9 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 202 without deviations which may have impact on the validity of the study. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are reliable:
Under the conditions used for the test, there was a significant immobilisation of the daphnias at concentrations >10 mg/L (definitive main test).
As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following EC-values were determined accordingly after 48 hours:
Mobility:
LOEC = 20.0 mg/L
NOEC = 10.0 mg/L
EC50 = 13.1 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 3.

Executive summary:

Isobutyl 4-chloro-3,5-diaminobenzoate was tested for acute toxicity towards daphnia according to OECD-Test Guideline 202 (version dated 13-Apr-2004) in compliance with GLP. In order to investigate the influence of the test item towards the daphnia, the swimming behaviour of the animals was recorded. Under the conditions used for the test, there was a significant immobilisation of the daphnia at concentrations >10 mg/L (main test). As a conclusion of the analytical part of this study, it can be stated that the concentrations of Isobutyl 4-chloro-3,5-diaminobenzoate remained sufficiently stable during incubation of 48hr: After 48 hr recoveries of >80% of the initial t₀-concentrations were measured in the treatments. Based on this, the reported effect concentrations should refer to nominal concentrations (geometric mean). The following EC-values (effective concentrations) were determined accordingly:

	Basis: Nominal Concentrations [mg/L]
	t24hr	t48hr
NOEC	10.0	10.0
LOEC	20.0	20.0
EC10	16.3	8.8
EC20	17.3	10.1
EC50	19.6	13.1

The test is considered valid as all conditions for validity were met.