Hexadecyltrimethylammonium methyl sulphate

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 25 September, 2012 to 28 September, 2012

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:

yes

Remarks:

- NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain constant pH and aid test chemical stability. - Chemical analyses was performed on parallel test vessels without algae in all test concentrations at the start and end of test

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling and preparation for analysis
Samples of 10 mL were taken at the start and end of the test at all concentrations (including parallel), in the stock solution and in the control. All samples were diluted 1:1 with leaching solution and then shaken in the leaching solution for approximately 10 minutes at 100 RPM before being further diluted as required (in leaching solution) and then filtered through 0.45 acrodisk filters (as required) before chemical analysis.(See annex 4 of the attached report for chemical analysis) in the attached background material.

Chemical analysis
All samples taken were analyzed following the method for chemical analysis. Validation of the analytical method was performed.

Vehicle:

no

Details on test solutions:

Preparation of solutions
A stock solution of 116 mg/L of the test substance was prepared by accurately weighing 0.0116 g of test substance, on an analytical balance. The testsubstance was then added to approximately 80 mL of the test media a homogeneous stock solution was achieved. The volume was made up to a 100 mL with the test media. The stock was kept stirring while in use to generate the test concentrations. Stock pH was 8.2. No pH adjustment was required. For the lowest 3 concentrations the stock was diluted a further 10 times to allow more accurate pipetting of the stock volumes.

Test concentrations
A range finding test was conducted using test solutions prepared as described above. Based on these results the following definitive nominal concentrations were prepared as previously detailed by adding the appropriate amounts of stock solution to test media so as to achieve the desired test concentration in a total volume of 40 mL in the test replicates. 0.005, 0.013, 0.031, 0.08 and 0.19 mg/L (inclusive of a non inoculated analytical parallel) were prepared. Six replicates without the test substance were also prepared as a control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata (freshwater unicellular algae (CCAP 278/4)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK
- Method of cultivation: Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase. For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Standard OECD medium (The de-ionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not more than 2.0 mg/L and a conductivity of less than 5 uS/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure).

Test temperature:

23 ± 2°C

pH:

6.8 - 7.6

Dissolved oxygen:

-

Salinity:

Standard OECD medium

Nominal and measured concentrations:

Nominal: 0.005, 0.013, 0.031, 0.08 and 0.19 mg a.i./L

Details on test conditions:

The test was carried out in a temperature-controlled illuminated orbital incubator in which the temperature was maintained at 23 ± 2°C. Uniform Illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 ± 0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 µE.m-2.s-1. The test vessels were agitated continuously at a speed sufficient to prevent sedimentation of the algae (100 rpm approx).

Preparation of the inoculum
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1*104 cells/mL in each of the test vessels.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate 99.5% purity lot H0234 JT baker

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.01 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.14 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.008 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.02 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Preliminary test
The preliminary study gave results indicating 100% effect at 0.1 mg/L and no effects at 0.01mg/L after 72 hours of exposure. This information was used to estimate the test concentration range for the definitive study.

Definitive test
During the definitive study all of the desired endpoints could be calculated and the required quality criteria for the test method were met. For all dose response data with the exception of the nominal concentration rate endpoint confidence limits could be calculated. ErC10and ErC50values could be calculated by a maximum likelihood probit plot. NOEC and LOEC were also determined using a Dunnetts test. Normal distribution and equality of variance were tested by the Shapiro-Wilks and the Bartlett’s tests. The data was indicated to be non normal and unequal in variance this can effect the accuracy of the Dunnetts test. Considering the overlap of the data at the NOEC with the control the study director considers the NOEC sufficiently reliable.

The ErC10 (72h) was calculated as 0.010 mg/L and the ErC50 (72h) was calculated as 0.14 mg/L (Growth rate) [Measure initial values]. The NOEC was calculated as 0.008 mg/L and the LOEC as 0.02 mg/L. (Growth rate) [measured initial values].

pH, temperature, light and purity of algae
The pH measurements show a maximum increase of 1.3 pH units in a control replicate and in some of the test concentrations. The temperature varied from 22.1 to 22.3 °C during the test, and the light intensity was 101.4 and 102.7 µmol·m-2·s-1 at the beginning and end of the test respectively. All measurements are in accordance with the required conditions described in the study plan and in the guideline. The five random samples checked at the end of the test were not contaminated with excessive levels of bacteria when inspected microscopically.

Chemical Analysis
Analytical quantification demonstrated an excellent initial level of the test substance in the stock solutions for both of the measured components. Theoretically therefore the correct amount of test substance was dosed to the test replicates. The measurements in the actual test replicates at the start of the test were in the region of 70% of the nominal concentrations. This reduction in concentration is typical of strongly adsorbing substances. The analytical measurements at the end of the test are around 40% of the nominal with the exception of the lowest concentration, which has a recovery of 70%. The latter result is unexplainably high as one would expect complete adsorption to algae and no recovery at such concentrations. With elevating concentration the recovery in comparison to the nominal does not increase as one may expect. This is due to the test substance being heavily influenced by the algae cells and progressively fewer cells being present at higher concentrations. In the absence of algae in the parallel vessels the C12 component remains practically unchanged from the measured initial values. The C14 component seems to be more heavily bound to the glass than the C12 component in the absence of algae cells. Due to the binding of the test material strongly to algae cells and the subsequent inhibition of algae growth the measured data in the test vessels at the end of the test is not considered an accurate representation of the exposure concentration and is more an indication of how much test substance can be removed from the algae cells sampled for analysis. The parallel samples indicate an excellent stability for C12 and some loss for C14 to the glass in the absence of algae. In the presence of algae this would likely not be the case as previously researched for surfactants in ref 4. The collected data clearly demonstrates the test substance to be strongly sorbing and therefore the end of test analytics are not truly representative of the realistic exposure. The endpoints will therefore be based on measured initial values (corrected for both components) as indicated in the test guideline as acceptable for algae studies with adsorbing substances at low concentrations. This is also supported by the C12 analytical data in the parallel vessels which demonstrated perfect stability during the study after 72 hours. Furthermore C12 is also the main component.

Quality criteria
The following quality criteria were met in the present study:
• The cell density of the controls increased at least a factor 16 within 72 h.
• The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%
• The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (documented as part of GLP laboratory maintenance).
• The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35 %

The following criterion was not met:
• The test substance concentration decreased below 80% of the nominal and initial concentrations during the test. This is not uncommon with adsorbing substances and is not in this case indicative of reduced exposure. Surfactant based substances are known to bind to algae cells (ref 4) actually stimulating complete exposure. This is however not measurable analytically as removing the test substance entirely from the algae cells making it available for analysis is not always achievable. This difficulty is acknowledged in the OECD test guideline (Ref 1). Use of the measured initial concentration is therefore considered acceptable for this test substance. Furthermore the continued inhibition of growth rate in later stages of the test indicates test substance presence.

The toxicity test and chemical analysis met all critical validity criteria required by the study plan, standard operating procedures and the test guideline. Analytical results of the initial concentrations were considered the most reliable for use in endpoint determination. This is due to the difficulty and inaccuracy of analytical measurement at the end of algae tests with adsorbing substances at low concentrations. The main C12 component showed excellent stability and little binding to glass also supporting the use of measure initial concentrations. All possible combinations of endpoint expression have been calculated in the tables and figures section should they be required by the regulatory authorities. The test can be considered an accurate representation of the effects of the test chemical to P.subcapitata at the concentrations tested.

Deviations from the study plan
• Geometric means only have not been used for endpoint determination as mentioned in the study plan. All possible methods (Nominal, measured mean, and measured initial) of expressing the endpoints have been calculated. Measured initial concentrations have been reported in the main body of the report as is considered acceptable according to the OECD 201 test guideline for adsorbing substances.
• The NOEC/LOEC calculations were conducted using the Dunnetts not the Williams test as indicated in the study plan. This is not detrimental to the study as both methods are acceptable in the study guideline.

Results with reference substance (positive control):

The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (documented as part of GLP laboratory maintenance). Hence, the quality criteria have been met in the present study.

Reported statistics and error estimates:

The mean values of the observed absorbance for each test substance concentration were used to calculate the yield and specific growth rate. Where possible, the EC10, 50, values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value cal¬culated for the specific growth rate is termed ErC50. The LOEC was determined by comparison of the growth at each concentration and the control using the Dunnett’s or William’s tests. The NOEC was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. All computations were performed using the TOXCALC version V 5.0.23 program and the validated Algal 2A spreadsheet (as per Ref 2 of the attached report). Endpoints were primarily expressed as measured initial concentrations as is acceptable for adsorbing substances in algae studies. Nominal and Geometric mean measured calculations were conducted also should these be required and are presented in the tables and figures section.

Results

Table 1a Chemical analysis results per component 

Sample mg/L(Nominal)	0h(mg/L) (C12)	72h(mg/L) (C12)	72hParallel(mg/L) (C12)
control	<LOD	<LOD	-
0.005	0.004	0.004	0.003
0.013	0.009	0.006	0.007
0.031	0.022	0.016	0.017
0.08	0.062	0.031	0.057
0.19	0.158	0.105	0.156
Stock 116	107.8	-	-
Samplemg/L (Nominal)	0h(mg/L) (C14)	72h(mg/L) (C14)	72hParallel(mg/L) (C14)
control	<LOD	<LOD	-
0.005	0.003	0.003	0.001
0.013	0.008	0.005	0.003
0.031	0.019	0.011	0.004
0.08	0.052	0.021	0.031
0.19	0.131	0.057	0.094
Stock 116	108.4	-	-

 LOD-limit of detection

- = not measured

 

Table 1b Chemical analysis results (corrected for AI) of each component

Sample mg/L(Nominal)	0h(mg/L)	% ofnominal	72h(mg/L)	% ofnominal	Geometric measured Meanmg/L	MeanRecoveryof  Nominal
control	<LOD	-	<LOD	-
0.005	0.0035	70	0.0035	70	0.0035	70
0.013	0.0084	64	0.0055	42	0.0068	52
0.031	0.0203	65	0.0140	46	0.0169	55
0.08	0.0569	71	0.0272	34	0.0394	50
0.19	0.145	76	0.0886	46	0.113	60

 

Correction for composition

C12 =50.0 % A.I. was corrected to 70.6% A.I as C12 and C14 represent 68% of the active component but were considered representative for the entire 96% of active ingredient as stated on the certificate of analysis in this case.

C14 =18.0 % A.I.was corrected to 25.2% A.I as C12 and C14 represent 68% of the active component but are considered representative for the entire 96% of active ingredient as stated on the certificate of analysis in this case.

Values in tables 1a were then corrected for composition as indicated and summed resulting in table 1b above.

A.I. = Active Ingredient.

Table 2 Raw data

Nominal Concentration	ABSORBANCE   Time (hours)				AUC	Inhibition %	SGR	Inhibition %
(mg/L)	0	24	48	72
Control	0.008	0.031	0.132	0.602	10.656		0.060
Control	0.008	0.035	0.202	0.753	14.244		0.064
Control	0.007	0.035	0.184	0.776	14.148		0.066
Control	0.008	0.034	0.166	0.746	13.272		0.063
Control	0.007	0.023	0.189	0.512	10.812		0.062
Control	0.009	0.035	0.171	0.550	11.004		0.058
Mean	0.008	0.032	0.174	0.657	12.356		0.062	0.0
0.005	0.005	0.033	0.172	0.755	13.680		0.064
0.005	0.005	0.034	0.172	0.716	13.236		0.063
0.005	0.005	0.036	0.190	0.667	13.128		0.062
Mean	0.005	0.034	0.178	0.713	13.348	-8.0	0.063	-1.0
0.013	0.005	0.032	0.145	0.564	10.716		0.060
0.013	0.005	0.032	0.159	0.670	12.324		0.062
0.013	0.005	0.034	0.172	0.663	12.600		0.062
Mean	0.005	0.033	0.159	0.632	11.880	3.9	0.061	1.8
0.031	0.005	0.023	0.017	0.050	1.260		0.023
0.031	0.005	0.033	0.122	0.475	9.120		0.057
0.031	0.005	0.034	0.123	0.447	8.832		0.056
Mean	0.005	0.030	0.087	0.324	6.404	48.2	0.051	18.5
0.08	0.005	0.031	0.072	0.195	4.512		0.043
0.08	0.005	0.030	0.078	0.215	4.872		0.045
0.08	0.004	0.031	0.120	0.177	5.508		0.046
					4.964	59.8	0.045	28.4
Mean	0.005	0.031	0.090	0.196
0.19	0.005	0.030	0.073	0.083	3.168		0.033
0.19	0.005	0.026	0.037	0.063	1.968		0.028
0.19	0.005	0.026	0.036	0.070	2.028		0.029
					2.388		0.030
Mean	0.005	0.027	0.049	0.072		80.7		51.2

 AUC= Area under curve (Biomass) SGR = Specific Growth Rate (Rate)

Table 3 Average specific growth rates and coefficients of variation during the whole test period in the control and section by section (Validity Criteria)

0-24	24-48	48-72	72h test mean	stdev	%CV	Mean %CV
0.056	0.060	0.063	0.0600	0.00341	5.68	14.83
0.061	0.073	0.055	0.0631	0.00921	14.60
0.067	0.069	0.060	0.0654	0.00481	7.36
0.060	0.066	0.063	0.0630	0.00291	4.62
0.050	0.088	0.042	0.0596	0.02470	41.44
0.057	0.066	0.049	0.0571	0.00872	15.27
		mean	0.0614
		stdev	0.0030
		CV%	4.8793

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the ErC10 (0-72 h) and ErC50 were determined to be 0.01 and 0.14 mg a.i./L (measured initial values), respectively. The NOEC for effects on algal growth and growth rate was determined to be 0.008 mg a.i./L (measured initial values).

Executive summary:

A study was conducted to determine the toxicity of the test substance, C12-14 ADMAES (96% active), to the algae Pseudokirchnerella subcapitata according to OECD 201 Guideline and EU Mehods C.3, in compliance with GLP. Algae were exposed to the test substance at nominal concentrations of 0.005, 0.013, 0.031, 0.08 and 0.19 mg/L. Algal cell concentrations were determined spectrophotometrically. All the validity criteria were fulfilled. Analytical assessment was performed for the test substance in aqueous samples, at the beginning, middle and at the end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). The test substance concentration decreased to below 80% of the nominal and initial concentrations during the test. This is because surfactant based substances are known to bind to algae cells actually stimulating complete exposure. This is however not measurable analytically as removing the test substance entirely from the algae cells, making it available for analysis is not always achievable. This difficulty is acknowledged in the OECD test guideline and a use of the measured initial concentration is therefore considered acceptable for this type of test substance. Under the study conditions, the ErC10 (0-72 h) and ErC50 were determined to be 0.01 and 0.14 mg a.i./L (measured initial values), respectively. The NOEC for effects on algal growth and growth rate was determined to be 0.008 mg a.i/L (measured initial values) (Kean, 2012).

Description of key information

Based on the results of the read across study, the 72 h ErC10 and ErC50 values of the read across substance, C12-14 ADMAES, for toxicity to algae was considered to be 0.01 and 0.14 mg a.i./L (measured initial values) respectively. The NOEC for effect on algal growth and growth rate was determined to be 0.008 mg a.i./L (measured initial values).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.14 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L

Additional information

A study was conducted to determine the toxicity of the read across substance, C12-14 ADMAES (96% active), to the algae Pseudokirchnerella subcapitata according to OECD 201 Guideline and EU Mehods C.3, in compliance with GLP. Algae were exposed to the read across substance at nominal concentrations of 0.005, 0.013, 0.031, 0.08 and 0.19 mg/L. Algal cell concentrations were determined spectrophotometrically. All the validity criteria were fulfilled. Analytical assessment was performed for the read across substance in aqueous samples, at the beginning, middle and at the end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). The read across substance concentration decreased to below 80% of the nominal and initial concentrations during the test. This is because surfactant based substances are known to bind to algae cells actually stimulating complete exposure. This is however not measurable analytically as removing the read across substance entirely from the algae cells, making it available for analysis is not always achievable. This difficulty is acknowledged in the OECD test guideline and a use of the measured initial concentration is therefore considered acceptable for this type of read across substance. Under the study conditions, the ErC10 (0-72 h) and ErC50 were determined to be 0.01 and 0.14 mg a.i./L (measured initial values), respectively. The NOEC for effects on algal growth and growth rate was determined to be 0.008 mg a.i/L (measured initial values) (Kean, 2012). Based on the results of the read across study, similar effect levels can be considered for the test substance.