N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Daphnids use for the test were not <24h; a mixture of juvenile and adult daphnids was exposed to the test item

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Carolina Biological Supply company, Burlington North carolina, USA
- Other: Adult Daphnia were transferred to a separate holding container. After one week, the large adults were removed, and the young Daphnia were maintained for an additional two weeks prior to testing; Culture medium was obtained by blending approximately 2 grams of alfalfa pellets with 3-4 dried brine shrimp per liter of water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Nominal and measured concentrations:

Control, 0.3, 0.9 and 3.0 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 15O-mL Griffin beakers, a final volume of 100-110 mL was used throughout the testing program

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

OTHER TEST CONDITIONS
- Adjustment of pH: 7.0 to 7.5
- Light intensity: flourescent lighting

EFFECT PARAMETERS MEASURED: Adult mortalities were recorded on a daily basis. Births observed during the experiment were noted but not enumerated, nor were mortalities among the newborn included in the daily record.

RANGE-FINDING STUDY: A pre-test was performed with the test item concentrations of 0, 0.18, 0.33, 0.58, 1.02 and 1.82 mg/L. After a test period of 96 h the EC50 was determined to be >1.8 mg/L. Based on these results the main test was performed with the test item concentration range of 0 - 3.0 mg/L.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Biological Results:

Mortality after 96 h: Control: 7.5 %; 0.3 mg/L: 50%, 0.9 mg/L: 63.6 %; 3 mg/L: 61.5 %

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Dec 2017 - 23 Oct 2018

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)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and the control
- Sampling method: Samples were analysed on the day of sampling; 1.8 mL from the approximate centre of the test vessels; Samples were taken at various time points (t=0, 1, 2, 4, 8, 24 and 48 h)
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg/L or 10 mg/L was stirred for 5-7 min with magnetic stirr. Afterwards the testitem was completely dissolve. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. Test solutions were clear and colorless up to 4.6 mg/L and increasingly pink colored at 10 and 100 mg/L at the end of the preparation procedure. It was noted that the test solution of 100 mg/L prepared for the combined limit/range-finding test darkened already during the first minutes after preparation.
- Controls: Only medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: More than two weeks old
- Feeding during test: No
- Food type: A suspension of fresh water algae
- Frequency: Daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel, renewal of culture: After 7 days of cultivation, half of the medium twice a week.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

CaCO3: 180 mg/L

Test temperature:

20 °C

pH:

8.0 - 8.1 (Control)
7.7 - 8.0 (Test item concentration)

Dissolved oxygen:

8.6 - 8.8 mg/L (Control)
8.3 - 8.9 mg/L (Test item concentration)

Nominal and measured concentrations:

Control, 0.46, 1.0, 2.2, 4.6 and 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel
- Type: open
- Material, size, headspace, fill volume: 60 mL, all-glass
- Aeration: No aeration of the test solutions was applied.
- No. of organisms per vessel: 20 per concentration
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L
- Culture medium different from test medium: Yes, a different medium was used for the test
- Others: Detail composition of test medium is included in section "Any other information on material and methods inncl tables"

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A daily photoperiod of 16 h
- Introduction of daphnids: Within 1 h after preparation of the test solutions

EFFECT PARAMETERS MEASURED: Immobility (including mortality): At 24 h and at 48 h; pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control; Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: One immobilised daphnid was observed in the control (5%) at the end of the test. No immobility was observed at the two lowest test concentrations of 0.10 and 1.0 mg/L, while all daphnids exposed to 10 and 100 mg/L were immobilised at the study end. Therefore, the expected EC50 was between nominally 1.0 and 10 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no mortality was observed

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of Daphnia magna was in agreement with the historical data.The 48h EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.

Reported statistics and error estimates:

The 24h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method. The 48h-EC50 could not be determined using a regression method. This was because there was no concentration between the highest concentration (A) at which 0% immobility and the lowest concentration (B) at which 100% immobility occurred. Instead, the EC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Test substance Nominal conc. (mg/L)
		Control	0.46	1.0	2.2	4.6	10
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	4	5
	B	0	0	0	0	5	5
	C	0	0	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	0	0	0	19	20
	Effect %	0	0	0	0	95	100
48	A	0	0	0	0	5	5
	B	0	0	0	0	5	5
	C	0	0	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	0	0	0	20	20
	Effect %	0	0	0	0	100	100

Table 2: Final Full Test: Test Samples (based on the hydrolysis product)

Time of sampling [hours]	Date of sampling	Date of analysis 1	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	16 Oct 2018	23 Oct 2018	0	n.d.	n.a.
			0.46	0.561	122
			1.0	1.21	121
			2.2	2.60	118
			4.6	5.21	113
			10	11.4	114
48	18 Oct 2018	23 Oct 2018	0	n.d.	n.a.	n.a.
			0.46	0.511	111	91
			1.0	1.15	115	95
			2.2	2.49	113	96
			4.6	5.04	109	97
			10	11.4	114	100

1) Samples were stored in the freezer (≤ -15°C) until the day of analysis.

n.d. Not detected.

n.a.  Not applicable.

Table 3: Effect Parameters

Parameter	Test substance Nominal conc. (mg/L)	95%-confidence interval  (mg/L)
24h-EC50	3.5	3.1-3.9
48h-EC50	3.2	2.2-4.6

Table 4: pH and Oxygen Concentrations (mg/L) During the Final Test

Test substance Nominal conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	8.8	8.0	8.6	8.1
0.46	8.8	7.9	8.5	8.0
1.0	8.9	7.9	8.3	8.0
2.2	8.9	7.9	8.4	8.0
4.6	8.7	7.8	8.4	7.9
10	8.5	7.7	8.4	7.9

Table 5: Validity criteria for OECD 202

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	no daphnids became immobilised or showed other signs of disease or stress	yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	the dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels	yes

Description of key information

EC50 (48h) 3.2 mg/L (Daphnia magna, OECD 202, nominal)

Additional information

One key study is available, in which the short term toxicity to invertebrates was investigated according to GLP and OECD guideline 202 (Migchielsen, 2018b). The test was performed with a test item concentration of 0.46, 1.0, 2.2, 4.6 and 10 mg/L (nominal) in a static test system with the test organism Daphnia magna. The initial measured content of the test item ranged between 113 and 122% and the aged measured content ranged between 109 and 115% of nominal. Therefore the results are given based on nominal concentrations. The EC50 (48 h) was determined to be 3.2 mg/L.

One additional study is available on the short-term toxicity of the test item to aquatic invertebrates (National Association of Photographic Manufacturers, 1977). The study was performed with the freshwater species Daphnia magna in a static test design. Daphnids were exposed to 0, 0.3, 0.9 and 3.0 mg/L of the test substance for a 96 h period. The LC50 (96 h) of the test item was determined to be 0.3 mg/L. However, the study was considered to be not reliable since the test design differs much from the OECD standrads (e.g. instead of neonates a mixture of juvenile and adults test organisms were used) .