4-amino-5-(ethylsulphonyl)-o-anisic acid

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February 2015 - 17 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of preregistration data requirements for Annex II (part A; Section 4) and Annex III (PART a; Section 5) of directive 91/414

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Acilian (4-Amino-5-ethylsulfonyl-2-methoxybenzoic acid)
- batch No.of test material:GS00012
- Expiration date of the lot/batch:February 28, 2015
- Purity test date:March 13, 2014
- Purity: 98.0 – 101.5% (nominal)
100.9% (analytical), according to certificate of analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature (20 ± 5 °C), in the dark
- Stability under test conditions: yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal concentration of the test item: 100mg/L.
- Sampling method:
Analysis of the Test Item Concentrations:
Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours). All samples were diluted by a factor of 2 with acetonitrile. One additional sample of the blank control and the dilution solvent was taken at test start and test end without any sample treatment.
The concentration of the test item was analysed in the duplicate test media samples from the only test concentration, and in the duplicate control samples, from both sampling times (0 and 48 hours).

Fortified Samples:Approximately 50 mg of the test item were dissolved (1 minutes ultrasonic treatment and 15 minutes stirring) in 50 mL test water to obtain a stock solution of approximately 1 g test item/L. Two stock solutions were prepared. Appropriate amounts of these stock solutions were diluted with test water to obtain fortified samples at a level of 40 and 140 mg test item/L. Exact values were documented in the raw data.

Standard Solutions used for the Quantification:
- Stock Solution:49.85 mg of the test item were dissolved in 50 mL acetonitrile to obtain a stock solution of approximately 1 g test item/L.
- Standard Solutions:Appropriate amounts of the stock solution were diluted with acetonitrile / test water (1/1, v/v) to obtain standard solutions in the range from 10 to 70 mg test item/L.
Analysis of treatment samples and control samples.
- Sample Preparation:The samples were allowed to thaw to room temperature.They were then shaken well and treated with ultrasound for 1 minute to obtain homogenous samples. The samples were already diluted with acetonitrile by factor 2 directly after sampling (see chapter 6.10). The samples were diluted further with acetonitrile / test water (1/1, v/v) to match the calibration range, if necessary.

- Sample storage conditions before analysis:All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.

The quantification of the test item Acilian was performed using liquid chromatography with UV detection.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the only test concentration of nominal 100 mg test item/L was prepared by dissolving 40 mg test item into 400 mL water by intense stirring for 30 minutes. The test media were prepared just before introduction of the daphnids (= start of the test).
- Controls:Reconstituted water - without addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the test media.Test medium was clear throughout the entire test duration.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: 5
- Source: Ibacon's in-house laboratory culture.
- Age of parental stock (mean and range, SD): From 2.5 to 18.25 hours old at test start.
- Feeding during test: The daphnids in the stock culture were fed at least on all working days.
- Food type: green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

ACCLIMATION
- Acclimation period: Not needed since since the test was performed in the same medium as the culturing

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Test organisms were considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20 °C at test start;
20 °C at test end

pH:

8.2 at test start;
8.0 at test end; and thus the pH-value did not vary by more than 1.5
units

Dissolved oxygen:

9.1 to 9.2 mg/L at test start;
8.9 to 9.0 mg/L at test end

Conductivity:

conductivity < 5 μScm-1

Nominal and measured concentrations:

Nominal : 100 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): closed,covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume:Glass, 100mL containing approximately 60 mL of test medium.
- Aeration: STIRRING
- 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:Reconstituted Water (Elendt "M4") was prepared according to OECD Test Guideline No 202
- Alkalinity:0.9 mmol/L
- Conductivity:< 5 μScm-1
- Culture medium different from test medium:No
- Intervals of water quality measurement: Start and end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h light / 8 h dark.
- Light intensity:The light intensity was 760 to 850 lux (measured once during the test).

EFFECT PARAMETERS MEASURED: EC50,EC10, NOEC and LOEC.
Number of immobile organisms after 24 and 48 hours -Test organisms were considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING TEST
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The pre-experiments were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no
After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of nominal 100 mg test item/L.
- Observations on body length and weight:No
- Other biological observations: No
- Mortality of control: No
- Other adverse effects control:No
- Abnormal responses:No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media.Test medium was clear throughout the entire test duration.

Results with reference substance (positive control):

- Results with reference substance valid : YES
EC50 [mg/L]:1.85 (24 HOURS) and 1.11 (48 HOURS)
EC20 [mg/L]: 1.68 (24 HOURS), 1.02 (48 HOURS)
EC10 [mg/L]: 1.60 (24 HOURS), 0.976 (48 HOURS)
NOEC [mg/L]: 1.0 (24 HOURS), 0.5 (48 HOURS): determinated from the raw data
LOEC [mg/L]: 2.0 (24 HOURS), 1.0 (48 HOURS): determinated from the raw data

Reported statistics and error estimates:

No statistical analysis was performed. The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Table 1. Results of the Determination of the Test Item in the Test Samples

Sample Description [mg test item/L]	AGE day	AGE hour	Concentration found [mg test item/L]^1	D.F.	Concentration calculated [mg test item/L]^1	Concentration nominal [mg test item/L]^2	% of nominal^2
Control (diluted)	0	0	<LOD	2	N.A.	0.000	N.A.
Control (diluted)	0	0	<LOD	2	N.A.	0.000	N.A.
Control (diluted)	2	48	<LOD	2	N.A.	0.000	N.A.
Control (diluted)	2	48	<LOD	2	N.A.	0.000	N.A.
100	0	0	52.733	2	105.466	100.000	105
100	0	0	52.528	2	105.056	100.000	105
100	2	48	53.138	2	106.275	100.000	106
100	2	48	52.235	2	104.470	100.000	104

mean value (n=4):105

RSD (n=4):1

1 The tabulated results represent rounded results calculated on the exact raw data

2 The results represent rounded values

LOD: Limit of Detection = 3.7 mg test item/L

n.a.: not applicable

RSD: Relative Standard Deviation

D.F.: Dilution factor

Table No.2: Results of the Determination of the Test Item in the Fortified Samples

Description	[mg test item/L]	Concentration found [mg test item/L]^1	D.F.	Concentration calculated [mg test item/L]^1	Concentration nominal [mg test item/L]^2		% of nominal^2
Analytical Blank Control	0	<LOD	2	n.a.	0.000		n.a.
Analytical Blank Control	0	<LOD	2	n.a.	0.000		n.a.
Fortified Sample	40	20.210	2	40.420	39.704		102
Fortified Sample	40	20.536	2	41.073	39.704		103
Fortified Sample	40	20.354	2	40.708	40.344		101
Fortified Sample	40	20.139	2	40.279	40.344		100
Fortified Sample	40	20.148	2	40.297	40.344		100
						mean value (n=5):		101
						RSD (n=5):		1
Fortified Sample	140	32.490	2	129.960	138.964		94
Fortified Sample	140	35.720	2	142.882	138.964		103
Fortified Sample	140	36.015	2	144.062	141.204		102
Fortified Sample	140	36.431	2	145.725	141.204		103
Fortified Sample	140	36.396	2	145.584	141.204		103
						mean value (n=5):		101
						RSD (n=5):		4

overall mean value (n=10):101

RSD (n=10):3

1 The tabulated results represent rounded results calculated on the exact raw data

2 The results represent rounded values

LOD: Limit of Detection = 3.7 mg test item/L

n.a.: not applicable

RSD: Relative Standard Deviation

D.F.: Dilution factor

Table No.3: Influence of Acilian on the Mobility of Daphnia magna

Nominal Concentration [mg test item/L]	No. of Daphnia tested	No. of immobilised Daphnia after		% of immobilised Daphnia after
		24h	48h	24h	48h
Control	20	0	0	0	0
100	20	0	0	0	0

Table No.4: Water Temperature in the Test Media at the Start and End of the Test

Nominal Concentration [mg test item/L]	Water temperatura (ºC)
	0 h	48 h
Control	20	20
100	20	20

Table No.5: Dissolved Oxygen Concentrations and pH-Values in the Test Media

Nominal Concentration [mg test item/L]	0 hours		48 hours
	pH	O2[mg/L]	pH	O2[mg/L]
Control	8.2	9.2	8.0	8.9
100	8.2	9.1	8.0	9.0

Table No.6: Appearance of Test Item in Test Medium

Nominal Concentration [mg test item/L]	Appearance of the Test Item in the Test Media
	0 h	24 h	48 h
100	0	0	0

Abbreviations:

0: No remarkable observations, clear test medium

1: Colouration caused by the test item

2: Turbidity caused by the test item

3: Inhomogeneous dispersion of the test item

4: Precipitation of the test item

5: Test item at the surface

6: Test item lying at the bottom of the test vessel

+: Effects were strongly pronounced

Effects in parenthesis were only slightly pronounced

Validity criteria fulfilled:

yes

Remarks:

The dissolved oxygen conc. at the end of the test should be ≥ 3 mg/L (≥ 8.9 mg O2/L obtained) of the air saturation value at the T used. Control immobilisation rate- not more than 10 % (0% obtained).

Conclusions:

No immobilized animal observed. The value EC50 (48h) is > 100 mg/L.

Executive summary:

The study was carried out with principles of GLP studies - OECD Guidelines 202 . The purpose of this study was to evaluate the influence of the test item Acilian on the mobility of Daphnia magna. This limit test was performed in compliance with the test guidelines to demonstrate that the test item has no toxic effect on daphnids up to at least this concentration. Twenty, young Daphnia magna (<24 h old, 5 per replicate) were exposed to either a control medium (test water) and 100 mg test item /L for 48 hours under static conditions. The immobility of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. There were no observed any immobilized animal and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid. The 48-hour NOEC was determined to be 100mg test item /L. The 48-hour LOEC was determined to be > 100 mg test item/.The EC50 (48h) was determined to be > 100 mg test item /L.

Description of key information

Key study: OECD 202, GLP study, No immobilized animal observed . The estimated 48-h EC50 is > 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Key study: The study was carried out with principles of GLP studies - OECD Guidelines 202. Twenty Daphnia magna (<24 h old, 5 per replicate) were exposed to a control medium and 100 mg test item /L for 48 hours under static conditions. The limit test was performed. The immobility of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. There were no observed any immobilized animal. The dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits. The 48-hour NOEC was determined to be 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L. The EC50 (48h) was determined to be > 100 mg test item/L.