Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl-1H-imidazole-1-ethanol and sodium hydroxide

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:

weight of evidence

Study period:

June 12, 1992 - January 7, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Study conducted equivalent to OECD 202 and GLP. Missing information on the age of the Daphnids. No accurate monitoring of the test concentrations and as there is no data reported about any results obtained with a reference substance, the sensitivity can not be checked. Also, missing information about number of animals at each test concentration and for the controls. Still regarded as a reliable study as the validity criteria can be verified and are fulfilled (and there is no definite data about the age of the Dahnids/about the sensitivity).

Qualifier:

according to guideline

Guideline:

other: DIN 38412, Teil 11

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

no data about the age of the Daphnids; no data about results obtained with a reference substance; no data about number of animals at each test concentration and for the controls and no accurate monitoring of the test concentrations

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The nominal concentrations are expressed based on an incorrect purity of the substance. These concentrations are here corrected:
- Concentrations: 325, 1250 and 7500 mg/L (aqueous solution), at t=0, t=24 and t=48h

Vehicle:

no

Details on test solutions:

The stock solution was prepared by adding 10 g of the testmaterial (aqueous solution) to 100 mL water.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: from own laboratory
No further details available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

No data

Test temperature:

20 - 21 degrees Celsius

pH:

At t=24h: 7.8-8.2
At t=48h: 8.0-8.1

Dissolved oxygen:

At t=24h: 83-94%
At t=48h: 85-94%

Salinity:

Not applicable

Nominal and measured concentrations:

The nominal concentrations are expressed based on an incorrect purity of the substance. These concentrations are here corrected:
Nominal concentrations: 0, 225, 325, 450, 625, 900, 1250, 1750, 2500, 7500 and 25000 mg/L (aqueous solution)
Nominal concentrations: 0, 78.75, 113.75, 157.5, 218.75, 315, 437.5, 612.5, 875, 2625 and 8750 mg/L (expressed as solid content)

Details on test conditions:

TEST SYSTEM
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to DIN 38412, Teil II

No further details available.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

255 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(aqueous solution)

Basis for effect:

mobility

Remarks on result:

other: Aqueous solution with a solid content of approximately 35%

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

89 mg/L

Nominal / measured:

nominal

Conc. based on:

other: expressed as solid content

Basis for effect:

mobility

Details on results:

- Based on the DOC measurements at t=0; t=24 and t=48 h, it is assessed that the concentrations remained >80% nominal (details not provided in this summary)
- The irrelevance of the DOC measurements is supported by the fact that the measurements at t=0 are higher than the nominal DOC values as well as by the fact that DOC values increase during the test (which is easily understandable as biological activity probably causes this increase).

Results with reference substance (positive control):

Not applicable

Validity criteria fulfilled:

yes

Conclusions:

In an acute Daphnia Magna study, conducted in accordance with DIN 38412, Teil II, an 48h-EC50 of 89 mg/L (expressed as solid content) was determined. It should be noted that the age of the Daphnids is not provided.

(1) Immobility in control < 10% (0%); 2) Oxygen >3 mg/L (>7.8 mg/L; reported as 85-94 %)

Executive summary:

In accordance with DIN 38412, Teil II, an acute toxicity study was conducted with Daphnia Magna. The test type was static and there was analytical monitoring. For the control and for the test groups, 2 replicates were used. A reference substance was not tested. The 48h-EC50 of the substance was determined to be 89 mg/L (expressed as solid content). All the validity criteria were fulfilled.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 July 2017 - 03 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:

15 December 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: 2.554

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 10 minutes.
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material: the test medium at 116.2 mg test item/L (45.5 mg solids/L) appeared to be slightly turbid and caused slight foaming. At 255.4 mg test item/L (100 mg solids/L), slight turbidity and foaming was observed.

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: 1-19 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:

20.5 to 20.6 °C at test start;
20.3 to 20.5 °C at test end

pH:

8.0 to 8.1 at test start;
7.9 at test end

Dissolved oxygen:

8.7 to 8.9 mg/L at test start;
8.2 to 8.4 mg/L at test end

Nominal and measured concentrations:

Nominal concentrations: 255.4, 116.2, 52.9, 24.0 and 11.0 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 84-100% of nominal values.

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: 220 to 500 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, 100% immobilisation was observed in the highest concentration while no immobility was observed in the lower concentrations. Therefore, the EC50 was expected to be between 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

64.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

25.4 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 nominal test item concentration of 24.0 mg test item/L (9.4 mg solids/L). At the nominal concentration of 52.9 mg test item/L (20.7 mg solids/L), 7 animals were immobile and 18 animals were immobile at the nominal concentration of 116.2 mg test item/L (45.5 mg solids/L). At the highest test concentration of 255.4 mg test item/L (100 mg solids/L), 20 daphnids were immobile (see table 2).
- At test end, the test medium appeared to be slightly turbid at 116.2 mg test item/L (45.5 mg solids/L) and turbid at 255.4 mg test item/L (100 mg solids/L)
- The 95% confidence interval for the EC50 was calculated to be 52.7 -79.6 mg/L (20.6 -31.2 mg solids/L)
- Analysis showed that the test concentrations remained 84-100% of nominal test concentrations. Therefore, the effect concentrations were based on nominal test concentrations.

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	0	0	9.591	1	9.591	11.000	87
11	0	0	9.943	1	9.943	11.000	90
11	2	48	9.051	1	9.051	11.000	82
11	2	48	9.774	1	9.774	11.000	89
						Mean value (n=4):	87
						RSD (n=4):	4
24	0	0	20.932	1	20.932	2400	87
24	0	0	20.212	1	20.212	2400	84
24	2	48	19.591	1	19.591	2400	82
24	2	48	20.309	1	20.309	2400	85
						Mean value (n=4):	84
						RSD (n=4):	3
52.9	0	0	50.787	1	50.787	52.900	96
52.9	0	0	50.447	1	50.447	52.900	95
52.9	2	48	45.703	1	45.703	52.900	86
52.9	2	48	34.483	1	34.483	52.900	^#65
						Mean value (n=4):	93
						RSD (n=4):	6
116.2	0	0	116.167	1	116.167	116.200	100
116.2	0	0	117.555	1	117.555	116.200	101
116.2	2	48	117.310	1	117.310	116.200	101
116.2	2	48	115.433	1	115.433	116.200	99
						Mean value (n=4):	100
						RSD (n=4):	1
255.4	0	0	126.955	2	253.909	255.400	99
255.4	0	0	125.225	2	250.449	255.400	98
255.4	2	48	118.792	2	237.583	255.400	93
255.4	2	48	122.773	2	245.546	255.400	96
						Mean value (n=4):	97
						RSD (n=4):	3
					Overall mean value (n=20)		92
					RSD (n=20)		7

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

LOD: Limit of Detection = 1 mg test item/L

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

^#considered to be an outlier, not used for calculation

Table 2 Influence of Miranol Ultra C32 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	20	0	0	0	0
24	20	0	0	0	0
52.9	20	1	7	5	35
116.2	20	2	18	10	90
255.4	20	1	20	5	100

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 for Miranol Ultra C32 towards Daphnia magna was 64.8 mg/L (25.4 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 Ultra C32 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, 24, 52.9, 116.2 and 255.4 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 84-100% of nominal test concentrations and thus the effect concentrations were based on nominal test concentrations.

In the lowest concentrations (11 and 24 mg/L) no daphnids became immobilised, while in the highest concentration all daphnids became immobilised. The 48h-EC50 of Miranol Ultra C32 was determined to be 64.8 mg/L (25.4 mg solids/L) with 95% confidence interval 52.7 -79.6 mg/L (20.6 -31.2 mg solids/L). All validity criteria were met and the study was considered to be valid.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

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.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The rationale to read across the data is attached in Section 13.

Reason / purpose for cross-reference:

read-across source

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: 220 to 500 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, 100% immobilisation was observed in the highest concentration while no immobility was observed in the lower concentrations. Therefore, the EC50 was expected to be between 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

64.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

25.4 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 nominal test item concentration of 24.0 mg test item/L (9.4 mg solids/L). At the nominal concentration of 52.9 mg test item/L (20.7 mg solids/L), 7 animals were immobile and 18 animals were immobile at the nominal concentration of 116.2 mg test item/L (45.5 mg solids/L). At the highest test concentration of 255.4 mg test item/L (100 mg solids/L), 20 daphnids were immobile (see table 2).
- At test end, the test medium appeared to be slightly turbid at 116.2 mg test item/L (45.5 mg solids/L) and turbid at 255.4 mg test item/L (100 mg solids/L)
- The 95% confidence interval for the EC50 was calculated to be 52.7 -79.6 mg/L (20.6 -31.2 mg solids/L)
- Analysis showed that the test concentrations remained 84-100% of nominal test concentrations. Therefore, the effect concentrations were based on nominal test concentrations.

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	0	0	9.591	1	9.591	11.000	87
11	0	0	9.943	1	9.943	11.000	90
11	2	48	9.051	1	9.051	11.000	82
11	2	48	9.774	1	9.774	11.000	89
						Mean value (n=4):	87
						RSD (n=4):	4
24	0	0	20.932	1	20.932	2400	87
24	0	0	20.212	1	20.212	2400	84
24	2	48	19.591	1	19.591	2400	82
24	2	48	20.309	1	20.309	2400	85
						Mean value (n=4):	84
						RSD (n=4):	3
52.9	0	0	50.787	1	50.787	52.900	96
52.9	0	0	50.447	1	50.447	52.900	95
52.9	2	48	45.703	1	45.703	52.900	86
52.9	2	48	34.483	1	34.483	52.900	^#65
						Mean value (n=4):	93
						RSD (n=4):	6
116.2	0	0	116.167	1	116.167	116.200	100
116.2	0	0	117.555	1	117.555	116.200	101
116.2	2	48	117.310	1	117.310	116.200	101
116.2	2	48	115.433	1	115.433	116.200	99
						Mean value (n=4):	100
						RSD (n=4):	1
255.4	0	0	126.955	2	253.909	255.400	99
255.4	0	0	125.225	2	250.449	255.400	98
255.4	2	48	118.792	2	237.583	255.400	93
255.4	2	48	122.773	2	245.546	255.400	96
						Mean value (n=4):	97
						RSD (n=4):	3
					Overall mean value (n=20)		92
					RSD (n=20)		7

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

LOD: Limit of Detection = 1 mg test item/L

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

^#considered to be an outlier, not used for calculation

Table 2 Influence of Miranol Ultra C32 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	20	0	0	0	0
24	20	0	0	0	0
52.9	20	1	7	5	35
116.2	20	2	18	10	90
255.4	20	1	20	5	100

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 for Miranol Ultra C32 towards Daphnia magna was 64.8 mg/L (25.4 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 Ultra C32 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, 24, 52.9, 116.2 and 255.4 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 84-100% of nominal test concentrations and thus the effect concentrations were based on nominal test concentrations.

In the lowest concentrations (11 and 24 mg/L) no daphnids became immobilised, while in the highest concentration all daphnids became immobilised. The 48h-EC50 of Miranol Ultra C32 was determined to be 64.8 mg/L (25.4 mg solids/L) with 95% confidence interval 52.7 -79.6 mg/L (20.6 -31.2 mg solids/L). All validity criteria were met and the study was considered to be valid.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The rationale to read across the data is attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

yes

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.

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

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

One short-term toxicity study to aquatic invertebrates with the registered substance is available (ref. Guhl 1993a), and 2 studies with structurally similar substances (Miranol BM and Miranol Ultra C32). The registered substance is Amphoacetates C12, while the structurally similar substances are Amphoacetates C12 -C14 and C8 -C18. The results of these studies can be read across to Amphoacetates C12 since the C12 alkyl chain is present in majority in those substances.

The Guhl (1993a) study with the registered substance is valid and reliable. The results of the other two studies are similar and the analytics of those studies confirm the analytical results of Guhl (1993a), i.e. it is scientifically justified to use nominal concentrations. Therefore, based on a weight-of-evidence approach it is considered justified to use the results of Guhl 1993a with Amphoacetates C12 as key result for the short-term toxicity to aquatic invertebrates endpoint.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

89 mg/L

Additional information

48h-EC50: 89 mg solids/L