Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 23, 2007 to July 30, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name:N-Alkyl(C12-C14)-N,N-dimethyl-N-ethylbenzylammonium chloride (ADEBAC)
Lot number:7226376
Expiry date:21 February 2008
Purity: 80.93% N-Alkyl(C12-C14)-N,N-dimethyl-N-ethylbenzylammonium chloride (ADEBAC), 9% ethanol, 1.6% free amine + Amine HCL
Appearance:Clear yellow liquid
Storage conditions:Room temperature in the dark
Stability: The a.i., ADEBAC, is hydrolytically and photolytically stable under the conditions of this study and related quaternary ammonium compounds have been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least seven years under standard laboratory conditions

Analytical monitoring:

yes

Details on sampling:

The concentrations of test substance in samples of the test cultures were measured during the definitive test using a LC-MS method of analysis. At the start of the definitive test, two samples (5 mL) were taken from the preparation vessels containing the remaining freshly-prepared control and test media. After samples were collected for cell counts at 72 h, the contents of replicate flasks for each group were pooled and a further two samples (5 mL) were taken for analysis. Additional samples were also taken from the 0.00277 and 0.0647 mg a.i./L flasks with no algal cells at 72 h, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells. On each occasion, the samples were placed in polypropylene tubes containing an aliquot (5 mL) of methanol containing formic acid (0.2%). One sample from each set was analysed and the other (reserve) sample was stored in a freezer in case further analysis was required. The reserve samples from Day 0 were analysed because the results obtained for the original set of samples were low. These low results were confirmed to be due to a calculation error. Following correction, the data were acceptable so the results for the reserved samples were not required and therefore have not been reported. On Day 3, analysis of the reserve samples was not considered necessary because the results for the original samples were considered acceptable. Thus, these reserve samples were discarded.

Vehicle:

yes

Remarks:

Water/Alcohol

Details on test solutions:

The test substance provided was a stock solution of 80% ADEBAC (nominally) in water/alcohol. Test solutions were prepared on the basis of the test substance as provided. However, this study was designed to determine the effects of the active ingredient (ADEBAC); therefore, throughout this report, the exposure concentrations and test results have been expressed in terms of active ingredient (ADEBAC). The method of test solution preparation used during the definitive test was based on the results of the range finding test. To minimise any loss of test substance by adsorption, all glassware used in the definitive test was pre-conditioned to the test substance at an appropriate concentration overnight before use. The pre-conditioning media were discarded immediately before the test solutions were freshly prepared. For the definitive test, an aqueous test preparation was made by dissolving the test substance (16 mg) in sterile algal nutrient medium (ca. 500 mL) in a volumetric flask. The contents of the flask were shaken before it was adjusted to volume with sterile dilution medium (2 L). Aliquots (0.855, 1.88, 4.13, 9.1 or 20 mL per 2 L) of this stock solution were diluted with sterile dilution medium to provide the test media. An aliquot (2.21 mL) of the secondary algal inoculum was added to a portion (400 mL) of the test medium, to give a nominal initial cell density of 1 x 104 cells/mL. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels. An aliquot (100 mL) of the remaining the test medium (without algal cells) was placed into empty, sterile vessels at 0.00277 and 0.0647 mg a.i./L and these flasks were incubated under test conditions and used for chemical analysis at the end of the test. The control cultures were prepared as for the test medium except that no test substance was added and a larger volume (700 mL) of medium was made.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Name
Pseudokirchneriella subcapitata, Strain No. SAG 61.81 (equivalent to CCAP 278/4). Unicellular green algae such as Pseudokirchneriella subcapitata are used as model systems in tests to determine whether a chemical affects the primary productivity of plants in the freshwater environment. Since many unicellular green algae have short generation times, this relatively short test can be used to assess the effects of a substance over many generations.

Source
Axenic, uni-cellular, liquid slope cultures of algae were obtained from Culture Collection of Sammlung Von Algenkulturen (SAG) at the University of Göttingen, Untere Karspüle, Göttingen, Germany and the slope used for the definitive test arrived on 2 July 2007.

Pre-culture
The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterized by a cell density of 1.81 x 106 cells/mL.

Culture medium
The algal pre-culture was maintained and the study was conducted in sterile nutrient medium as recommended in OECD Procedure 201 and Directive 92/69/EEC Official Journal No. L383A Part C.3. The water used to prepare this medium was purified reverse osmosis water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.5-23.9°C

pH:

7.36-9.04

Nominal and measured concentrations:

0 (negative control), 0.00277, 0.00608, 0.0134, 0.0295 and 0.0647 mg a.i./L nominal test substance concentrations

Details on test conditions:

Experimental design and test concentrations
The study comprised two range finding tests, an analytical trial and a definitive test. The first range finding test employed nominal concentrations of 0.000809, 0.00809, 0.0809 and 0.809 mg a.i./L and an algal dilution medium control group, each with three replicate vessels per exposure group. After 72 h, algal growth had been inhibited at 0.0809 and 0.809 mg a.i./L compared to the control. At 0.000809 and 0.00809 mg a.i./L growth was inhibited by 25 and 43% compared to the control. No results were obtained for analytical samples taken during the range finding test due to a problem with the original analytical method. Following the development of a suitable analytical method, a trial was performed in which a batch of test medium was prepared at nominal 0.00809 mg a.i./L.  Analysis of a sample of this medium confirmed that the intended exposure concentration could be adequately achieved (84% of nominal). A second range finding test was then conducted at nominal concentrations of 0.000809 and 0.00809 mg a.i./L as the inhibition noted in the first range finding test was unexpected. An algal dilution medium control group was also included in the test and for each group, three replicate vessels were employed. After 72 h, no adverse effects on algal growth were observed. The definitive test employed five concentrations plus an algal nutrient medium control group. Based on the result of the range finding tests, the following nominal ADEBAC concentrations of 0.00277, 0.00608, 0.0134, 0.0295 and 0.0647 mg a.i./L. In the definitive, six flasks were established and incubated for the control group and three flasks for each test group, plus an additional flask at nominal concentrations of 0.00277 and 0.0647 mg a.i./L, which contained test medium but no algal cells. Media remaining in the preparation flasks were used to measure water quality and test concentrations at the start of the test. Before the start of the test, the required number of empty test vessels (250 mL conical flasks), were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (100 mL), each flask was then loosely plugged with a foam bung.

Measurement of growth
Samples were taken from control and test media at 0, 24, 48 and 72 h to monitor algal growth. At the start of the definitive test, a sample (2 mL) was removed from the control and test media remaining in the preparation flasks and diluted with Isoton (18 mL). Cell densities of the resultant samples were measured, using a Coulter Z Series Particle Count and Size Analyser, to verify that the correct initial cell density of nominal 1 x 104 cells/mL had been achieved. At 24, 48 and 72 h, one sample (2 mL) was removed from each of the control and test flasks containing algal cells and analysed as outlined at 0 h using the particle counter. The cell numbers estimated using the particle counter were based on the mean of three consecutive counts that were corrected for background counts of uninoculated dilution medium. The presence of any abnormal cells was also noted during screening of each test level.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

other: EbC50

Remarks:

Area under the growth curve

Effect conc.:

ca. 0.019 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence intervals - 0.0169 & 0.021 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Area under the growth curve and growth rate

Effect conc.:

ca. 0.006 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Remarks:

Average specific growth rate

Effect conc.:

ca. 0.026 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Intervals - 0.0261 & 0.027

Remarks:

mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Area under the growth curve and growth rate

Effect conc.:

ca. 0.013 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

EC10 Cell density at 72 h:
ErC10 = 0.0162 mg a.i./L (0.015 to 0.0176 mg a.i./L)
Biomass at 72 h:
EbC10 = 0.0111 mg a.i./L (0.0093 to 0.0131 mg a.i./L)

EC50 Cell density at 72 h:
ErC50 = 0.0265 mg a.i./L (0.0261 to 0.027 mg a.i./l)
Biomass at 72 h:
EbC50 = 0.0189 mg a.s./l (0.0169 to 0.021 mg a.i./L)

Results

The measured concentrations of test substance ranged between 77 and 110% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved. After 72 h, the measured levels at 0.00277 to 0.0134 mg a.i./L were maintained, with values ranging between 84 and 94% of their starting values. At the two highest test levels (0.0295 and 0.0647 mg a.i./L), the measured levels had decreased to 46 and 66% of their starting values, respectively. The intended exposure concentrations were adequately achieved and maintained. In addition, the test substance is known to be readily soluble and hydrolytically stable, and since the test vessels were pre-treated with test substance to prevent loss of the test substance to absorption/binding to the vessel walls, any 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). Therefore, effect concentrations were determined based on nominal concentrations of the active ingredient. After 72 h, analysis of samples of medium nominally containing test substance at 0.00277 mg a.i./L, which had been incubated without algal cells, showed a decrease in the measured level from 110% to 65% of its nominal value whereas at 0.0647 mg a.i./L, the medium incubated without algal cells showed that the achieved concentration had been maintained.

Algal growth

Individual cell densities for each culture and the mean values were calculated. The calculated area under the growth curve and average specific growth rate values were expressed in terms of percentage inhibition relative to growth in the control cultures.

	Test substance (mg a.i./L;nominal)
Area under the growth curve (72 h)	0.0111 (0.0093 & 0.0131)
EbC10 value (95% Confidence Intervals)
EbC50 value (95%Confidence Intervals)	0.0189 (0.0169 & 0.021)
Lowest observed effect concentration (LOEbC)	0.0134
No observed effect concentration (NOEbC)	0.00608
Average specific growth rate (0 -72 h)
ErC10 value (95% Confidence Intervals)	0.0162 (0.015 & 0.0176)
ErC50 value (95% Confidence Intervals)	0.0265 (0.0261 & 0.027)
Lowest observed effect concentration (LOErC)	0.0134
No observed effect concentration (NOErC)	0.00608

Mean cell density of control cultures at 0 hours: 0.804 x 10⁴cells/mL

Mean cell density of control cultures at 72 hours: 232 x 10⁴cells/mL

Observations

No microscopic abnormalities of the cells were detected.

Environmental parameters

Water quality (temperature and pH) remained within acceptable limits throughout the study. Continuous monitoring of the incubator showed that temperature ranged between 22.2 and 23.8°C during the definitive test. Measurements of light intensity ranged between 6670 and 7640 lux during the definitive test and individual values were within the range -8 and +5. On the day of preparation, the test media were colourless.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, after 72 h of exposure to test substance, the EbC50 and ErC50 values, were determined to be 0.0189 and 0.0265 mg a.i./L, respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were determined to be 0.00608 mg a.i./L and 0.0134 mg a.i./L respectively, for both area under the growth curve and growth rate.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, C12-14 ADEBAC (active: 80.93%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Three replicate algal cultures, with a nominal initial cell density of approximately 1 x 104cells/mL, were exposed to test substance at nominal concentrations of 0.00277, 0.00608, 0.0134, 0.0295 and 0.0647 mg a.i./L in OECD algal medium for 72 h, under static conditions. The test vessels were pre-exposed to dilutions of test substance to prevent loss of the test substance by absorption/binding to the vessel walls. One untreated control group with six replicates containing algal cell inoculum and no test substance were also prepared. The exposure levels of test substance in aqueous samples of test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially acheived and maintained the test results were expressed in terms of their nominal values. Cell numbers were counted daily to monitor growth. The test results were expressed in terms of the area under the growth curve and average specific growth rate. ErC50 value (95% Confidence Intervals) for average specific growth rate was found to be 0.0265 (0.0261 & 0.027) mg a.i./L, whereas, EbC50 value (95% Confidence Intervals) for area under the growth curve was found to be 0.0189 (0.0169 & 0.021) mg a.i./L. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were 0.00608 mg a.i./L and 0.0134 mg a.i./L respectively, for both area under the growth curve and growth rate. Under study conditions, after 72 h of exposure to test substance, the EbC50 and ErC50 values, were determined to be 0.0189 and 0.0265 mg a.i./L, respectively. The NOEC and the LOEC values were determined to be 0.00608 mg a.i./L and 0.0134 mg a.i./L respectively, for both area under the growth curve and growth rate (Jenkins, 2008).

Description of key information

The 72 h EbC50, ErC50, NOEC and LOEC values for the test substance for toxicity to freshwater green algae, were determined to be 0.0189, 0.0265, 0.00608 and 0.0134 mg a.i./L, respectively.​

Key value for chemical safety assessment

EC50 for freshwater algae:

0.026 mg/L

EC10 or NOEC for freshwater algae:

0.006 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C12-14 ADEBAC (active: 80.93%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Three replicate algal cultures, with a nominal initial cell density of approximately 1 x 104cells/mL, were exposed to test substance at nominal concentrations of 0.00277, 0.00608, 0.0134, 0.0295 and 0.0647 mg a.i./L in OECD algal medium for 72 h, under static conditions. The test vessels were pre-exposed to dilutions of test substance to prevent loss of the test substance by absorption/binding to the vessel walls. One untreated control group with six replicates containing algal cell inoculum and no test substance were also prepared. The exposure levels of test substance in aqueous samples of test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially acheived and maintained the test results were expressed in terms of their nominal values. Cell numbers were counted daily to monitor growth. The test results were expressed in terms of the area under the growth curve and average specific growth rate. ErC50 value (95% Confidence Intervals) for area under the growth curve was found to be 0.0265 (0.0261 & 0.027) mg a.i./L, whereas, EbC50 value (95% Confidence Intervals) for average specific growth rate was found to be 0.0189 (0.0169 & 0.021) mg a.i./L. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were 0.00608 mg a.i./L and 0.0134 mg a.i./L respectively, for both area under the growth curve and growth rate. Under study conditions, after 72 h of exposure to test substance, the EbC50 and ErC50 values, were determined to be 0.0189 and 0.0265 mg a.i./L, respectively. The NOEC and the LOEC values were determined to be 0.00608 mg a.i./L and 0.0134 mg a.i./L respectively, for both area under the growth curve and growth rate (Jenkins, 2008).