Quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl)trimethyl, chlorides

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:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study was conducted similar to OECD guideline 202.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

yes

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Because of the very rapid removal of the test substance from the water column, the geometric mean of the 0, 24 and 48-h concentrations was considered to be the overall exposure concentration in the acute toxicity tests.

Hardness:

Laboratory blended water (total hardness ~150 mg/L)
Southwest well water (total hardness ~350 mg/L)
River water (total hardness ~300-350 mg/L)

Nominal and measured concentrations:

Please refer to the table 1 of the attached background material for details on nominal and measured concentrations of all three test water types.

Details on test conditions:

Three water types were used in this test: laboratory blended water (total hardness ~150 mg/L), Southwest well water (total hardness ~350 mg/ L) and river water (total hardness ~300-350 mg/L).

The river water, exemplifying a natural surface water that received sewage effluent, was collected from the White River (Indiana) and transported for cold storage (~4°C).

Acute toxicity tests of 48-h duration were conducted in each water type employing seven concentrations of test substance plus control and an isopropanol (IPA) control. The tests were repeated to ensure reproducibility. The same procedures were utilized in the repeat tests with the exception of testing a more recently collected batch of White River water. There was no renewal of test waters throughout the 48-h test period. Mortality was recorded daily and water chemistry measurements were taken at the beginning and conclusion of the test period for control waters only. Each test substance concentration was verified by radiochemical counting of triplicate 10 mL samples collected from the fresh stock solution (0 h) and from a randomly selected beaker after 24 and 48 h.

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 17.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: In Southwest well water (LC50 values in the initial and repeat tests were 19.8 and 15.3 μg/L, respectively).

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 12.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: In blended water (LC50 values in the initial and repeat tests were 16.3 and 8.8 μg/L, respectively).

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 98.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: In river water (initial test result only).

Details on results:

In the initial acute toxicity tests, little difference existed between LC50 values for blended and well waters. The river water LC50, however, was 5 to 11 times higher, possibly related to the presence of solids causing test substance adsorption and reduced bioavailability. An additional factor may have been that the river water contained endogenous nutritional sources perhaps enhancing daphnid resistance to the effects of the test substance.

Please refer to the table 2 of the attached background material for mortality data.

Reported statistics and error estimates:

The 48-h LC50 values were determined by probit analysis based on the geometric mean of the 0, 24 and 48-h concentrations to reflect overall exposure concentrations.

Validity criteria fulfilled:

not specified

Conclusions:

Under the test conditions, the 48 h LC50 measured concentration were 17.5 μg a.i./L in Southwest well water, 12.6 μg a.i./L in blended water and 98.9 μg a.i./L in river water

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance, C16-18 and C18-unsatd. TMAC (48.4% active in water, radiochemical purity: 98%) to Daphnia magna according to a method similar to OECD Guideline 202, under static conditions. Twenty Daphnids were exposed to seven nominal concentrations of the test substance in three different water types (i.e., laboratory blended water, Southwest well water and river water). The nominal concentration in Southwest well and blended water were 11.5, 15.5, 21.0, 28.0, 37.0, 49.0 and 65.0 µg/L and 115, 155, 210, 280, 370, 490 and 650 µg/L in river water. The tests were repeated to ensure reproducibility. The same procedures were utilized in the repeat tests with the exception of testing a more recently collected batch of river water. Mortality was recorded daily and water chemistry measurements were made at the beginning and end of the test period for control waters only. Each test substance concentration was verified by radiochemical counting of triplicate 10 mL samples collected from the fresh stock solution (0 hour) and from a randomly selected beaker after 24 and 48 h. Because of the very rapid removal of the test substance from the water column, the geometric mean of the 0, 24 and 48-hour concentrations was considered to be the overall exposure concentration in the acute toxicity tests. The initial and repeat 48-h LC50s values of the test substance based on the measured concentrations were determined to be 19.8 and 15.3 μg/L (geometric mean: 17.5 μg/L) in Southwest well water, 16.3 and 8.8 μg/L (geometric mean: 12.6 μg/L) in blended water and 98.9 μg a.i./L (initial) in rive water respectively. In the initial acute toxicity tests, little difference existed between LC50 values for blended and well waters. The river water LC50, however, was 5 to 11 times higher, possibly related to the presence of solids causing test substance adsorption and reduced bioavailability. An additional factor may have been that the river water contained endogenous nutritional sources perhaps enhancing daphnid resistance to the effects of the test substance. Under the test conditions, the 48 h LC50 measured concentration were 17.5 μg a.i./L in Southwest well water, 12.6 μg a.i./L in blended water and 98.9 μg a.i./L in river water (TRS-HPV, 2001).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From February 22, 2007 to February 24, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Two samples (5 mL) were taken from the freshly-prepared control and test media. After 48h, the contents of the test vessels from each group were pooled and further samples (5 mL) were taken for analysis. The samples were taken using a pipette and placed in glass vials; both of these items were pre-treated with a silanising agent (DMDCS) before use.

On each occasion, one of the samples was analysed and the other was stored in case further analysis was required. The reserve samples were stored frozen; it was not considered necessary to analyse these samples since the results obtained for the original samples were considered acceptable. Thus, the reserve samples were discarded.

Details on test solutions:

Preparation of test solutions:
The test organisms were maintained and the tests were conducted in softened Elendt M4 medium. The medium was prepared in deionised water produced by reverse osmosis. A stock solution for preparation of the test substance concentrations was prepared by diluting an aliquot of the test substance (19.6 mg) in the dilution medium (0.5 L) in a volumetric flask (1L). The contents of this flask were shaken vigorously before it was adjusted to volume with dilution medium. Aliquots (0.7, 1.2, 1.9, 3.1 or 5.5mL) of this stock solution were diluted with dilution medium to provide the test media.
- Mean water quality parameters, including temperature, pH, dissolved oxygen, total hardness and alkalinity remained within acceptable limits throughout the study.

Monitoring of the test substance concentration:
Duplicate samples of each freshly prepared control and test media were collected and the concentration of the test substance measured using an LC-MS method of analysis. The measured concentrations of the test substance ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was determined using nominal concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

Original Daphnia broodstock was obtained from the Institute National de Recherche Chimique Appliqué (IRChA), France. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8L of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2ºC. A photoperiod of 16h light: 8h dark was maintained, with 60-min transition periods of subdued lighting at the beginning and end of each light phase. The light intensity of the test area was 611 lux.

- The culture medium was renewed three times each week.

-Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given (0.05 to 0.08 mg carbon per daphnid).
- Culture conditions ensure that the stock animals reproduce by pathenogenesis.
- The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by gravid adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24h old, were used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 1ºC

pH:

7.44-7.73

Dissolved oxygen:

99-104% air saturation value

Nominal and measured concentrations:

Nominal concentration: 0.007, 0.012, 0.019, 0.031, 0.052 mg a.i./L.

Details on test conditions:

Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L. The Daphnia were placed at random into glass vessels, four replicates of five animals per vessel, each containing approximately 150 mL of medium (resulting in a loading rate of 30 mL medium per organism). Each vessel was loosely covered with a watch glass. The temperature of the test area was 20 ± 1ºC. A 16-hour light: 8-hour dark photoperiod was maintained, with 60-minute periods of subdued lighting at the beginning and end of each phase. No supplementary aeration was employed and no feed was given during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.023 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.019-0.24 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.0144-0.0177 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.031 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Test substance analysis:
The measured concentration of the test substance ranged between 80-97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e., representative of exposure of the organism to the nominal concentrations) and the EC50 value was determined using nominal concentrations.
Immobility: See the above results under 'Effect concentrations' and the table under 'any other information on results incl. tables'.

Results with reference substance (positive control):

The results from the most recent test performed prior to this study indicated that the reference substance 4h EC50 to Daphnia magna was 0.52 mg/L. This was within the range typically obtained in the laboratory (0.3 to 0.8 mg/L)

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal concentrations. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Immobilisation data

Concentration (mg a.i./l)	Percent Immobile at:
	24 h	48 h
0.0	0	0
0.007	0	0
0.012	0	5
0.019	15	85
0.031	100	100
0.052	100	100

For further details on all measurements, please refer to the attachment under 'Attached bakground material'.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48h EC50 value of the source substance for the immobilisation of Daphnia magna was 0.016 mg a.i./L and the 48h NOEC was 0.012 mg a.i./L. The 48h EC50 for the target substance is calculated as 0.012 mg/L upon correction for MW.

Executive summary:

A study was conducted to determine the short-term toxicity of the source substance, C12 -16 ADBAC (Purity: 51%) to Daphnia magna according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L under static conditions. Duplicate samples of each freshly prepared control and test media were collected and the concentration of test substance was measured using an LC-MS method of analysis. The measured concentrations ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48 hours, the measured levels ranged between 30 and 42% of nominal. The source substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the substance to prevent loss of the substance to absorption/binding to the vessel walls. Therefore, the reduction of substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was therefore determined using nominal concentrations. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007). The 48h EC50 for the target substance is calculated as 0.012 mg/L upon correction for MW.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

From 11 March 1988 to 13 March 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

A clear stock solution of 3.03 mg/mL was prepared by diluting 0.0303 g of the test substance with distilled water in a 10 mL flask. The test solutions were prepared by adding the appropriate volume of stock solution to dilution water to total 1,000 mL. Each solution was mixed on a magnetic stirrer for 30 seconds and then divided into 4 beakers to provide replicate exposure treatments, each containing 200 mL. The remaining 200 mL of test solution were used for 0 h water quality measurement.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Name of vehicle: Water
- Concentration of vehicle in test medium (stock solution): Stock solution of 3.03 mg/mL was prepared by diluting 0.0303 g of the test substance with distilled water in a 10 mL flask

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna
- Source: Springborn Life sciences, Inc., Wareham, Massachusetts
- Age at study initiation (mean and range, SD): ≤24 h
- Feeding during test: No

ACCLIMATION
- Acclimation period: Cultures of the test organism over many generation has been continuously maintained at the lab
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Solution of green algae and yeast suspension daily
- Feeding frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L as CaCO3

Test temperature:

20-21⁰C

pH:

7.9-8.3

Dissolved oxygen:

98-99%

Nominal and measured concentrations:

0, 236, 394, 667, 1,091, 1,818 and 3,030 µg/L as nominal concentration

Details on test conditions:

The test was conducted in 250 mL glass beaker. A clear stock solution of 3.03 mg/mL was prepared by diluting 0.0303 g of the test substance with distilled water in a 10 mL flask. The test solution was prepared by adding the appropiate volume of stock solution to dilution water to a total volume of 1 L. Each solution was mixed on a magnectic stirrer for 30 seconds and then divided into 4 beakers to provide replicate exposure treatments, each containing 200 mL. The remaining 200 mL of test solution was used for 0 hour water quality measurement and then discarded. One set of 4 control beakers was prepared containing the same dilution water and maintained under the same conditions as the exposure concentration, but without the test subsatnce. The ambient air temperature was controlled in order to maintain test solution temperature at 20 ± 1⁰C. The test solution was not aerated. The test area was illuminated with Sylvania GRO-LUX and cool white fluorescent lights at an intensity of 70 footcandles at the solution surface.

See below tables for detials.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

550 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

280 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: i.e., equivalent to 92.4 µg a.i. /L)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 236 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, behavioural and physical abnormalities

Remarks on result:

other: i.e., equivalent to 78 µg a.i. /L)

Details on results:

The 48 h EC50 and 95% confidence interval (calculated by probit analysis) was 280 (i.e. 92.4 ug a.i./L). The NOEC was <236 µg/L (<78 ug a.i./L), the lowest tested concentration.

Table 1: Immobilisation data

Test­-Substance Concentration (nominal) [µg a.i./l]
	ImmobileDaphnia
	Number      24 h         48 h		Percentage       24 h        48 h		Oxygen [mg/l] 48 h	pH   48 h	Tempera­ture [°C] 48 h
0	0	0	0	0	8.4	8.4	not known
79	0	4	0	20	8.9	8.5
131	1	19	5	95	9.0	8.5
222	17	20	85	100	9.0	8.5
364	20	20	100	100	-	-
606	20	20	100	100	-	-
1010	20	20	100	100	-	-

 

Table 2: Effect data

	EC50^1	95 % c.l.	EC0^1	EC100^1
24 h [µg a.i./l]	183 (n)	130-223	-	-
48 h [µg a.i./l]	93 (n)	83-100	< 79 (n)	-

¹indicate if effect data are based on nominal (n) or measured (m) concentrations

 

Table 3: Validity criteria for acute daphnia immobilistaion test according to OECD Guideline 202

	fulfilled	Not fullfilled
Immobilisation of control animals <10%	YES
Control animals not staying at the surface	YES
Concentration of dissolved oxygen in all test vessels >3 mg/l	YES
Concentration of test substance³80% of initial concentration during test	*

* Not known for no analyses were performed

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 48 h EC50 and NOEC of the source substance were 280 and <236 µg/L, (i.e., equivalent to 0.09 and <0.08 mg a.i./L), respectively

Executive summary:

A study was conducted to determine the acute toxicity study of the source substance, TMAC C (33% active in water), to the Daphnia magna according to a method similar to OECD Guideline 202, in compliance with GLP. Daphnia magna (10 per dose) were exposed to the sources substance at nominal concentrations of 0, 236, 394, 667, 1,091, 1,818 and 3,030 µg/L for 48 h under static conditions and mortalities were determined. No analytical determination of the read across substance was performed. Throughout the test period no visible sign of undissolved material was observed in the exposure solutions. Immobilisation was determined after 24 and 48 h. Through-out the 48-h exposure all organisms which were not immobilized appeared normal as compared to the control daphnids i.e., behavior and physical appearance. The 48 h EC50 and 95% confidence interval were calculated by probit analysis as 280 (250 - 300) µg/L. The NOEC was < 236 µg/L, the lowest tested concentration. Under the study conditions, the 48 h EC50 and NOEC were 280 and <236 µg/L (nominal; i.e., equivalent to 0.093 and <0.08 mg a.i./L), respectively (Surprenant, 1988).  

Description of key information

In line with the biocides assessment report and as a conservative approach, the same 48 h EC50 value of 0.012 mg a.i./L (nominal) based on read across from C12-16 ADBAC has been considered further for hazard/risk assessment. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

12 µg/L

Additional information

Study 1: A study was conducted to determine the short-term toxicity of the source substance, C12 -16 ADBAC (Purity: 51%) to Daphnia magna according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L under static conditions. Duplicate samples of each freshly prepared control and test media were collected and the concentration of test substance was measured using an LC-MS method of analysis. The measured concentrations ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48 hours, the measured levels ranged between 30 and 42% of nominal. The source substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the substance to prevent loss of the substance to absorption/binding to the vessel walls. Therefore, the reduction of substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was therefore determined using nominal concentrations. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007). The 48h EC50 for the registered substance is calculated as 0.012 mg/L upon correction for MW.

Study 2:  A study was conducted to determine the acute toxicity study of the source substance, TMAC C (33% active in water), to the Daphnia magna according to a method similar to OECD Guideline 202, in compliance with GLP. Daphnia magna (10 per dose) were exposed to the source substance at nominal concentrations of 0, 236, 394, 667, 1,091, 1,818 and 3,030 µg/L for 48 h under static conditions and mortalities were determined. No analytical determination of the source substance was performed. Throughout the test period no visible sign of undissolved material was observed in the exposure solutions. Immobilisation was determined after 24 and 48 h. Through-out the 48-h exposure all organisms which were not immobilized appeared normal as compared to the control daphnids i.e., behaviour and physical appearance. The 48 h EC50 and 95% confidence interval were calculated by probit analysis as 280 (250 - 300) µg/L. The NOEC was < 236 µg/L, the lowest tested concentration. Under the study conditions, the 48 h EC50 and NOEC were 280 and <236 µg/L (nominal, i.e., equivalent to 0.093 and <0.08 mg a.i./L), respectively (Surprenant, 1988).  

Study 3: A study was conducted to determine the short-term toxicity of the test substance, TMAC T (48.4% active in water, radiochemical purity: 98%) to Daphnia magna according to a method similar to OECD Guideline 202, under static conditions. Twenty Daphnids were exposed to seven nominal concentrations of the test substance in three different water types (i.e., laboratory blended water, Southwest well water and river water). The nominal concentration in Southwest well and blended water were 11.5, 15.5, 21.0, 28.0, 37.0, 49.0 and 65.0 µg/L and 115, 155, 210, 280, 370, 490 and 650 µg/L in river water. The tests were repeated to ensure reproducibility. The same procedures were utilized in the repeat tests with the exception of testing a more recently collected batch of river water. Mortality was recorded daily and water chemistry measurements were made at the beginning and end of the test period for control waters only. Each test substance concentration was verified by radiochemical counting of triplicate 10 mL samples collected from the fresh stock solution (0 hour) and from a randomly selected beaker after 24 and 48 h. Because of the very rapid removal of the test substance from the water column, the geometric mean of the 0, 24 and 48-hour concentrations was considered to be the overall exposure concentration in the acute toxicity tests. The initial and repeat 48-h LC50s values of the test substance based on the measured concentrations were determined to be 19.8 and 15.3 μg/L (geometric mean: 17.5 μg/L) in Southwest well water, 16.3 and 8.8 μg/L (geometric mean: 12.6 μg/L) in blended water and 98.9 μg a.i./L (initial) in river water respectively. In the initial acute toxicity tests, little difference existed between LC50 values for blended and well waters. The river water LC50, however, was 5 to 11 times higher, possibly related to the presence of solids causing test substance adsorption and reduced bioavailability. An additional factor may have been that the river water contained endogenous nutritional sources perhaps enhancing daphnid resistance to the effects of the target substance. Under the test conditions, the 48 h LC50 measured concentration were 17.5 μg a.i./L in Southwest well water, 12.6 μg a.i./L in blended water and 98.9 μg a.i./L in river water (TRS-HPV, 2001).

The biocide assessment report available from RMS Italy on TMAC C (ECHA assessment report, 2016), considered study no. 2 to be supportive because the endpoints were based on nominal concentrations, which could underestimate the toxicity. However, similar to the fish endpoint, several available reliable source studies with DDAC and BKC, also indicated toxicity in the same range. From the Lonza Cologne GmbH dossier, the RMS retrieved a read across data to DDAC as 48h EC50 = 0.062 mg a.s./L, calculated as 48h EC50 = 0.047 mg a.s./L upon correction for MW, and from the Nouryon Surface Chemistry AB dossier, a read across from C12-16-BKC (or C12-16 ADBAC) of 48h EC50 = 0.016 mg a.s./L, calculated as 48h EC50 = 0.012 upon correction for MW. The two read across endpoints were considered to be equally reliable, but the RMS selected the read across data to C12-16 ADBAC with the lowest 48 h EC50 = 0.016 mg a.i./L, as the worst case and calculated the 48 h EC50 for TMAC T at 0.012 mg a.i./L by applying correction for MW.   

Therefore, in line with the biocides assessment report and as a conservative approach, the same 48 h EC50 value of 0.012 mg a.i./L based on read across to C12-16 ADBAC has been considered further for hazard/risk assessment.