Isobutyl 4-chloro-3,5-diaminobenzoate

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:

2016-06-25 - 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Version dated 23-March-2006

Deviations:

yes

Remarks:

see below "Any other information on materials and methods incl. tables"

GLP compliance:

yes (incl. QA statement)

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: An aliquot of the algal stock culture was diluted with test medium to an absorbance value of E578 nm = 0.015 of the algal suspension giving an initial algal cell concentration of 3*10exp3 cells/mL. The determination of cell concentration was performed by photometrical measurement using a calibration curve prepared by using a spectrophotometer at 578 nm. The test item was prepared as follows: Separate solutions (see below) were made for each test concentration in ultrapure water. Aliquots of these solutions were made up with ..intermediate dilution" (see section above) and algal inoculum (or diluted ..intermediate dilution") to give 50 mL of final volume.
Prior to use in the test the test item was milled in a mortar in order to get fine particles of the test item which could be better dissolved in the aqueous phase hereafter. The test item was introduced into the test solutions as follows: First, suspensions of the test item were individually prepared for each test concentration in ultrapure water by shaking on a shaking machine for 1.0 hr at approx. 130 rpm followed by 2*10 minutes of ultrasonication (screening test; main test 1*10 min) and by stirring for 1 hr on a magnetic stirrer (screening test; main test 0.5 hr). Thereafter the suspensions were transferred into 8 test vessels (screening test) or 10 test vessels, respectively (main test). By the addition of the "intermediate dilution" and the algal inoculation the final nominal concentrations were achieved.
- Controls: medium without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Strain-No. 86.81
- Age of inoculum (at test initiation): Three days prior to starting the test, a pre-culture was inoculated using the algal stock culture.
- Method of cultivation: The algae were cultivated under aseptic / sterile conditions.

ACCLIMATION
- Acclimation period: Three days before starting of the test (main test), an algal pre-culture was made which was inoculated from an algal stock culture derived from the algal type culture collection Göttingen in order to guarantee sterile conditions at all.
- Culturing media and conditions (same as test or not): The pre-culture was incubated under the same conditions as the test solutions to be tested afterwards.
- Any deformed or abnormal cells observed: none stated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

25.2 to 26.3°C* (measured with a data logger [Elpro Ecolog])

pH:

8.04 - 9.85

Nominal and measured concentrations:

Nominal: 0, 5, 10, 20, 40, 80 mg/L
Found (t0): 0, 4.575, 8.609, 16.404, 31.452, 74.622 mg/L
Found (t72h): 0, 4.687, 8.570, 16.152, 29.823, 64.644 mg/L
Geometric mean measured concentration (0-72h): 0, 4.63, 8.59, 16.28, 30.63, 69.45 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: cylinders
- Material, size, headspace, fill volume: heat-sterilized glass, 50 mL final volume
- Initial cells density: 3*10E3
- Control end cells density: 9.1*10E5
- No. of vessels per concentration (replicates): 10 (main test) / 8 (screening)
- No. of vessels per control (replicates): 10 (main test) / 8 (screening)

GROWTH MEDIUM
- Standard medium used: yes, but the concentration of NaHC03 was increased to be twice of that indicated in the Guideline 201, as this concentration has been found to be optimal.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Mineral nutrient salts:
1.5 g NH4CI 13F130032 VWR
1.2 g MgCI2 x 6 H20 A799733043 Merck
1.8g CaCI2 x 2 H20 A0263182203 Merck
1.5g MgS04 x 7 H20 A0320286223 Merck
0.16g KH2P04 A0761277513 Merck
were dissolved in 1 L deionised water (abbreviated "Dl-H20" hereafter) and autoclaved

FeCI3- Solution:
0.064 g FeCI3x6H20 15D270031 VWR
0.10 g Na2EDTA x 2 H20 K45714718432 Merck
was dissolved in 1 L Dl-H20 and used freshly on the day of preparation.

Trace Elements:
185 mg H3B03 A0724165431 Merck
415 mg MnCI2x4H20 A0536427427 Merck
3 mg* ZnCI2 B0974516344 Merck
1.5 mg* CoCI2x6H20 B0744239219 Merck
0.01 mg* CuCI2 x 2 H20 K45082633421 Merck
7 mg* Na2Mo04 x 2H20 A0751921520 Merck
were dissolved in 1 L Dl-H20 and autoclaved. To include components marked "*", solutions were prepared by an additional dilution step using 1 mL of 300 mg ZnCI2/100 mL Dl-H20, 1 mL of 150 mg CoCI2*6 H2O/100 mL Dl-H20, 100 uL of 10 mg CuCI2 * 2 H2O/100 mL Dl-H20, and 2 mL of 350 mg Na2Mo04 * 2 H2O/100 mL Dl-H20.

Sodium Hydrogen Carbonate:
1.00 g/L NaHC03 K 44832529410 Merck
was introduced in the solid form directly into the "intermediate dilution" which then was sterilized by ultrafiltration. The final concentration of NaHC03 was twice as concentrated as indicated in the OECD Guideline 201.

Ultrapure water generating: Purelab Classic D1, USF Seral; Ser.-No. 1470984-10 01 (ELGA Labwater)

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous
- Light intensity and quality: illumination rate of > 120 uE/m2s ([=<8000 Lux]

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer, daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (main test), 10 (screening test)
- Range finding study: 0, 1, 10, 100 mg/L
- Test concentrations: 0, 5, 10, 20, 40, 80 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Results should be based on the nominal concentrations applied

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

both growth rate and yield

Remarks on result:

other:

Remarks:

Results should be based on the nominal concentrations applied

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none stated
- Unusual cell shape: none stated
- Colour differences: none stated
- Flocculation: not stated
- Aggregation of algal cells: not stated
- Any stimulation of growth found in any treatment: an activation of 12.4% was determined for the parameter yield at the lowest main test concentration of 5.0 mg/L compared to the control
- 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):

not required

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 201 with no relevant deviations (only minor deviation with no influence on the integrity of the study), the results were so obtained via a scientifically reasonable method. The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. Hence, there is no doubt that the obtained results are reliable: The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L (screening test), and the main test was performed using five concentrations in the range of 5 to 80 mg/L.
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 72hr: After 72 hr there was a recovery of 86.6 to 102.4% of the initial concentration measured at time t0. Recoveries of the nominal concentrations were in the range of 74.6 to 93.7%. Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.
The following EC-values (72h; growth rate; nominal concentrations) were determined accordingly:
LOEC 5.0 mg/L
NOEC 10.0 mg/L
EC50 = 21.9 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:

The toxicity of Isobutyl 4-chloro-3,5-diaminobenzoate towards algae was tested according to OECD-Guideline No. 201 under GLP, in the Version dated 23-March-2006. The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L (screening test), and the main test was performed using five concentrations in the range of 5 to 80 mg/L. The following EC-values were obtained:

On the Basis of the nominal Concentrations [mg test item/ L]

Yield (0 - 72 hr)                                                              EC10           7.1            95%-CL   lower           6.1                             upper           8.2                                            EC20           8.4            95%-CL   lower           7.3                             upper            9.7                                           EC50           11.6            95%-CL   lower            9.7                             upper            13.8                               Yield    LOEC           10.0                             NOEC          5.0 Section-by-section growth rate (0-72hr)                              EC10          14.1            95%-CL   lower            6.4                             upper           30.9                                            EC20           16.4            95%-CL   lower            7.4                             upper            36.4                                EC50          21.7            95%-CL   lower           7.4                             upper           59.4                                                     LOEC           20.0                            NOEC           10.0

Growth rate (0 - 72 hr)                                                     EC10            13.7            95%-CL   lower           12.9                             upper           14.6                               EC20            16.1            95%-CL   lower            15.1                             upper            17.2                               EC50            21.9            95%-CL   lower            20.0                             upper             23.7       Growth rate    LOEC           10.0                              NOEC             5.0

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 72hr: After 72 hr there was a recovery of 86.6 to 102.4% of the initial concentration measured at time t₀. Recoveries of the nominal concentrations were in the range of 74.6 to 93.7%.

Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.

The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. The increase of pH in the control by 1.77 units fails the requirements of the Guideline OECD 201 but is not considered to have an influence on the integrity of the results being due to good growth of the algae.