Miristalkonium chloride

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 24, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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-benzylammonium chloride (ADBAC C12-C14)
Lot number: 612212
Expiry date: 21 December 2009
Purity: 99.1% C14 ADBAC, 0.6% Free amine, 0.4% Amine hydrochloride
Appearance: White powder
Storage conditions: Room temperature
Stability: The a.i., ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five 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 at 72 h from the flasks containing test substance at 0.00342 and 0.08 mg/L, but with no algal cells, 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. One sample from each set was analysed and the other sample was stored in a freezer in case further analysis was required. This was necessary for the samples of control medium and test medium at 0.00342 mg/L taken on Day 0. A measurable level of test substance was found in the original control sample and the result for the sample taken at 0.00342 mg/L was slightly high, at 139% of nominal. The reserve sample taken at 0.00342 mg/L on Day 3 was not analysed even though the result for the original sample was high (176% of nominal) because it was obviously erroneous given the level measured at Day 0 and the lack of an effect observed at this level. All of the remaining reserve samples were discarded because the results obtained for the original samples were considered acceptable.

Vehicle:

yes

Remarks:

OECD algal medium

Details on test solutions:

Test solution preparation
The test solutions were not prepared on the basis of the percent active ingredient. 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 (16.2 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 (1.75 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 0.00342 and 0.08 mg/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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 2.28 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:

22.3-24.2°C

pH:

7.49-9.23

Nominal and measured concentrations:

0 (negative control), 0.00342, 0.00751, 0.0165, 0.0364 and 0.08 mg/L nominal test substance concentrations

Details on test conditions:

Experimental design and test concentrations
The study comprised a range finding test, an analytical trial and a definitive test. The range finding test employed nominal concentrations of 0.001, 0.01, 0.1 and 1 mg/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 0.1 mg/L (100%) compared to the control; no adverse effects on cell growth were noted at 0.001 or 0.01 mg/L. 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 0.01 mg/L. Analysis of a sample of this medium confirmed that the intended exposure concentration could be adequately achieved (90% of nominal). The definitive test employed five concentrations plus an algal nutrient medium control group. Based on the result of the range finding test, the following nominal concentrations of ADBAC C12-C14 were employed: 0.00342, 0.00751, 0.0165, 0.0364 and 0.08 mg/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 0.00342 and 0.08 mg/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: ErC50

Effect conc.:

ca. 0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.0191 & 0.0208 mg/L

Key result

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

ca. 0.008 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 0.00718 & 0.00866 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.003 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.008 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

EC10 Cell density at 72 h:
ErC10 = 0.00886 mg/L (0.00784 & 0.0099 mg/L)
Biomass at 72 h:
EbC10 = 0.00373 mg/L (0.00299 & 0.00452 mg/L)

EC50 Cell density at 72 h:
ErC50 = 0.0199 mg/L (0.0191 & 0.0208 mg/L)
Biomass at 72 h:
EbC50 = 0.00785 mg/L (0.00718 & 0.00866 mg/L)

Results

The measured concentrations of test substance ranged between 80 and 139% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved. A low but measurable level of the test substance (0.00379-0.00404 mg/L) was found in the control samples at the start of the test; the samples were thought to have been contaminated during either sampling or processing as no test substance was found in the sample analysed at the end of the definitive test. After 72 h, the measured concentrations had been adequately maintained (between 84 and 108% of their starting values) except at 0.0165 mg/L where the level had decreased to 70% of its starting value. This demonstration of test concentration stability excludes the high level (176%) measured at 0.00342 mg/L, which was also thought to have been contaminated after removal from the test flasks. No adverse effects on cell growth were noted at this level whereas at the next highest level (0.00751 mg/L), where the measured level was comparable to that found in the sample at 0.00342 mg/L, cell growth was significantly reduced. Consequently, the analytical result at 0.00342 mg/L was considered to be erroneous. 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. After 72 h, analysis of samples of medium containing test substance at 0.00342 and 0.08 mg/L, which had been incubated without algal cells, were lower (57 and 67% of nominal) than test media incubated in the presence of algal cells.

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/L; nominal)
Area under the growth curve (72 h)
EbC10 value (95% Confidence Interval)	0.00373 (0.00299 & 0.00452)
EbC50 value (95% Confidence Interval)	0.00785 (0.00718 & 0.00866)
Lowest observed effect concentration (LOEbC)	0.00751
No observed effect concentration (NOEbC)	0.00342
Average specific growth rate (0 -72 h)
ErC10  value (95% Confidence Interval)	0.00886 (0.00784 & 0.0099)
ErC50 value (95% Confidence Interval)	0.0199 (0.0191 & 0.0208)
Lowest observed effect concentration (LOErC)	0.00751
No observed effect concentration (NOErC)	0.00342

Mean cell density of control cultures at 0 h: 1.31 x 10⁴cells/mL

Mean cell density of control cultures at 72 h: 254 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, although the pH of the control and test cultures increased by more than 1.5 pH units during the 72 h exposure period. The increase in pH observed during the definitive test was attributed to the high level of cell growth that occurred but is not thought to have affected either the validity or integrity of the study because the validity criteria for this type of test were met by the control cultures. Continuous monitoring of the incubator showed that temperature ranged between 22.3 and 24.2°C during the definitive test. Measurements of light intensity ranged between 7020 and 8070 lux during the definitive test and individual values were within the range -8 and +6. On the day of preparation, the test media were colourless.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, after 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.00785 and 0.0199 mg/L, respectively. The NOEC and LOEC values for both the parameters were determined to be 0.00342 mg/L and 0.00751 mg/L respectively (Jenkins, 2008).

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, C14 ADBAC (purity: 99.1%), 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 0.00342, 0.00751, 0.0165, 0.0364 and 0.08 mg/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 achieved and were maintained between renewals of the media, 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. The 72 h ErC50 and ErC10 values (95% Confidence Intervals) for average specific growth rate were found to be 0.0199 (0.0191 & 0.0208) and 0.00886 (0.00784 & 0.0099) mg/L, whereas, 72 h EbC50 and EbC10 values (95% Confidence Intervals) for area under the growth curve were found to be 0.00785 (0.00718 & 0.000866) and 0.00373 (0.00299 & 0.00452) mg/L respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were 0.00342 mg/L and 0.00751 mg/L respectively, for both area under the growth curve and growth rate. Under study conditions, the 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.00785 and 0.0199 mg/L, respectively. The NOEC and LOEC values for both the parameters were determined to be 0.00342 mg/L and 0.00751 mg/L respectively (Jenkins, 2008).

Description of key information

Based on the study results, the 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.00785 and 0.0199 mg/L, respectively. The NOEC and LOEC values for both the parameters were determined to be 0.00342 mg/L and 0.00751 mg/L respectively (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.02 mg/L

EC10 or NOEC for freshwater algae:

0.003 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C14 ADBAC (purity: 99.1%), 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 of0.00342, 0.00751, 0.0165, 0.0364 and 0.08 mg/Lin 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 achieved and were maintained between renewals of the media, 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. The 72 h ErC50 and ErC10 values (95% Confidence Intervals) for average specific growth rate were found to be 0.0199 (0.0191 & 0.0208) and 0.00886 (0.00784 & 0.0099) mg/L, whereas, 72 h EbC50 and EbC10 values (95% Confidence Intervals) for area under the growth curve were found to be 0.00785 (0.00718 & 0.000866) and 0.00373 (0.00299 & 0.00452) mg/L respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were 0.00342 mg/L and 0.00751 mg/L respectively, for both area under the growth curve and growth rate. Under study conditions, the 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.00785 and 0.0199 mg/L, respectively. The NOEC and LOEC values for both the parameters were determined to be 0.00342 mg/L and 0.00751 mg/L respectively (Jenkins, 2008).