Acetic acid, 2-chloro-, reaction products with 2-C11-13-alkyl-4,5-dihydro-1H-imidazole-1-ethanol and sodium hydroxide

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:

18 July 2017 - 16 August 2017

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:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

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 pre-registration 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)

Remarks:

13 March 2017

Specific details on test material used for the study:

Correction factor for purity: 2.63

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: at t=0 duplicate samples were taken from the freshly prepared test media, at t=48 duplicate test samples were taken from the test media by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: all samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test item was applied individually, directly to the test medium. Subsequently, the test solutions were stirred for 15 minutes.
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material: the test item caused foaming in the test media of 54.4, 120 and 263 mg test item/L (20.7, 45.5 and 100 mg solids/L). Additionally, at a concentration of 263 mg test item/L (100 mg solids/L) the test medium was observed to be slightly turbid.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnia
- Clone: 5
- Source: in-house laboratory stock
- Age at study initiation: 1-21 hours old
- Age of parental stock: not indicated
- Feeding during breeding: daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of the test facility.
- Feeding during test: no

ACCLIMATION: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The daphnids were bred in the laboratories of the test facility under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg CaCO3/L

Test temperature:

19.9 to 20.5 °C at test start;
20.4 °C at test end

pH:

7.9 to 8.1 at test start;
7.9 at test end

Dissolved oxygen:

9.2 to 10.0 mg/L at test start;
8.6 to 8.8 mg/L at test end

Nominal and measured concentrations:

Nominal concentrations: 263, 120, 54.4, 24.7 and 11.3 mg test item/L (equivalent to 100, 45.5, 20.7, 9.4 and 4.3 mg solids/L)
Measured concentrations: see table 1 in 'any other information on results'
Mean measured concentrations were 79-97% of nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of 100 mL, fill volume: 60 mL, covered with a lid
- 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'')
- Culture medium different from test medium: no
- Intervals of water quality measurement: water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 160 to 270 lux

EFFECT PARAMETERS MEASURED: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, no immobility was observed in any of the concentrations tested. Therefore the EC50 was expected to exceed the highest tested concentration.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

177 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

67.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: solid fraction

Basis for effect:

mobility

Details on results:

- Abnormalities observed: no
- After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 120 mg test item/L (45.5 mg solids/L). At the highest test concentration of 263 mg test item/L (100 mg solids/L), 20 daphnids were immobile (see table 2).
- At test end, the test medium in the highest concentration of nominal 263 mg test item/L (100 mg solids/L) was observed to be slightly turbid.
- Analysis showed that the test concentrations remained 79-97% of nominal test concentrations. For the lowest concentration, the measured values at t=48 were below 80% of the nominal concentration, however, the nominal values were used for calculating the effect concentrations. This was done because the NOEC is 120 mg test item/L and the recoveries in the higher concentration range show nominal recoveries in the freshly prepared and aged test samples.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 48h-EC50: 1.06 mg/L
- Other: based on the 24h-EC50 of 1.48 mg/L, the sensitivity was according to the proposed OECD levels (24h-EC50 between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1 Measured concentrations

Sample description			Concentration found	D.F.	Concentration calculated	Concentration nominal
(mg test item/L)	Sampling day	Age (h)	(mg test item/L)*	1	(mg test item/L)*	(mg test item/L)	% of nominal*
Control	0	0	<LOD	1	n.a.	0.000	n.a.
Control	0	0	<LOD	1	n.a.	0.000	n.a.
Control	2	48	<LOD	1	n.a.	0.000	n.a.
Control	2	48	<LOD	1	n.a.	0.000	n.a.
11.3	0	0	10.153	1	10.153	11.300	90
11.3	0	0	9.954	1	9.954	11.300	88
11.3	2	48	7.761	1	7.761	11.300	69
11.3	2	48	7.645	1	7.645	11.300	68
						Mean value (n=4):	79
						RSD (n=4):	15
24.7	0	0	22.645	1	22.645	24.700	92
24.7	0	0	22.197	1	22.197	24.700	90
24.7	2	48	22.286	1	22.286	24.700	90
24.7	2	48	22.230	1	22.230	24.700	90
						Mean value (n=4):	90
						RSD (n=4):	1
54.4	0	0	51.625	1	51.625	54.400	95
54.4	0	0	53.194	1	53.194	54.400	98
54.4	2	48	51.910	1	51.910	54.400	95
54.4	2	48	52.049	1	52.049	54.400	96
						Mean value (n=4):	96
						RSD (n=4):	1
120	0	0	115.866	1	115.866	120.000	97
120	0	0	116.251	1	116.251	120.000	97
120	2	48	117.327	1	117.327	120.000	98
120	2	48	115.440	1	115.440	120.000	96
						Mean value (n=4):	97
						RSD (n=4):	1
263	0	0	110.633	2	221.266	263.000	84
263	0	0	116.767	2	233.534	263.000	89
263	2	48	113.672	2	227.345	263.000	86
263	2	48	107.259	2	214.517	263.000	82
						Mean value (n=4):	85
						RSD (n=4):	4
					Overall mean value (n=20)		89
					RSD (n=20)		10

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

LOD: Limit of Detection = 2 mg test item/L

n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

italics: The values are below 80% of the nominal concentration, however, the nominal values were used for calculating the effect concentrations. This was done because the NOEC is 120 mg test item/L and the recoveries in the higher concentration range show nominal recoveries in the freshly prepared and aged test samples.

Table 2 Influence of Miranol BM Conc. on the mobility of Daphnia magna

Nominal concentration (mg test item/L)	No. of Daphnia tested	No. of immobilized Daphnia		% of immobilized Daphnia
		After 24 h	After 48 h	After 24 h	After 48 h
Control	20	0	0	0	0
11.3	20	0	0	0	0
24.7	20	0	0	0	0
54.4	20	0	0	0	0
120	20	0	0	0	0
263	20	2	20	10	100

Validity criteria fulfilled:

yes

Remarks:

See 'overall remarks' section

Conclusions:

The 48h-EC50 for Miranol BM Conc towards Daphnia magna was 177 mg/L (67.3 mg solids/L).

Executive summary:

A short term toxicity test was performed, according to OECD guideline 202 and GLP principles, to assess the potential of Miranol BM Conc to induce immobility in Daphnia magna. The test item was corrected for the solid fraction and added to the medium at nominal concentrations of 11.3, 24.7, 54.4, 120 and 263 mg test item/L (corresponding to 4.3, 9.4, 20.7, 45.5 and 100 mg solids/L). Twenty daphnia per concentration and a blank control, divided over four vessels, were exposed to the test solutions for 48 hours. The number of immobilised daphnids after 24 hours and 48 hours was recorded. Duplicate samples for analysis were taken at t=0 and t=48 to determine actual exposure concentrations. Analysis showed that the test concentrations remained 79 -97% of nominal test concentrations. The effect concentrations were based on nominal test concentrations since the measured concentration was below 80% of initial in the lowest concentration only and no effects were seen at this concentration.

Only in the highest test concentration an effect was observed. In the other test concentrations no daphnids became immobilised while in the highest test concentration 100% of the introduced daphnids became immobilised. The 48h-EC50 of Miranol BM Conc. was determined to be 177 mg/L (67.3 mg solids/L). All validity criteria were met and the study was considered to be valid.

Description of key information

Two studies with the registered substance (mono-acetate form and di-acetate form) are available, as well as 5 older studies with a substance analogue. The EC50 values for the registered substance are 67.4 mg solids/L and >100 mg solids/L. Comparing the older data with the results of the daphnia studies performed in 2017 show that the newer studies give a higher EC50 value. This is expected to be related to the degree of purity of the substances tested. The results from the acute studies with daphnids are considered more relevant and reliable than the older reported EC50 values. See the read-across report in Section 13 for full details of the read-across rationale. The lowest EC50 value obtained with the registered substance (67.3 mg/L based on solid content for the mono-acetate form) is used as a worst-case key value for chemical safety assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

67.3 mg/L

Additional information

C12 -C14 mono-acetate:

EC50: 67.3 mg/L; NOEC 45.5 mg/L

C12 -C14 di-acetate:

EC50: >100 mg/L; NOEC 20.7 mg/L