Quaternary ammonium compounds, benzyl-C16-18-alkyldimethyl, chlorides

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 September, 2012 to 16 November, 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

Analytical monitoring:

yes

Details on sampling:

To ensure that the test substance was quantified as completely as possible in this study all concentrations were measured in the test vessels and in an additional parallel replicate at the start and at the end of the first test. Furthermore in the second study extraction of the glass surfaces of the test vessels in the region of effect and total analysis of the full 40mL of the test vessels with two different extraction fluids was conducted in order to gain as much information as possible about the test substance behavior in the test system.

Sampling and preparation for analysis
For the first study samples of 10 mL were taken at the start and end of the test at all concentrations (including parallel where present), 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 for chemical analysis report.

For the second study 10 mL samples at the start of the study were taken in the same manner as described above but at the end of the study the entire replicate was sacrificed for analysis via the addition of 40 mL leaching solution or acidified leaching solution to the test vessels. Furthermore the test vessels at 0.057 and
0.15 mg/L were extracted with leaching solution to establish the glass bound fraction of the test substance. 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.


Chemical analysis
All samples taken were analyzed following the method detailed in annex 4 of the attached background materail for chemical analysis) in the attached background material. Validation of the analytical method is also presented there.

Vehicle:

no

Details on test solutions:

Preparation of solutions
A stock solution of 105 mg/L and 54mg/L of the test substance was prepared in the first and second studies respectively by weighing accurate amounts of the test substance, on an analytical balance. The test chemical was then added to approximately 80 mL of the test media a homogeneous stock solution was achieved after ultrasonication. This resulted in slight warming of the stock solution to approximately 34 ºC. The volume was then made up to a 100 mL with the test media. During the first study the stock was then diluted a further 10 times to allow accurate pipetting of the volumes required to make test concentrations. The stock was kept stirring while in use. Due to the absence of a parallel replicate in the second study each replicate was prepared with 60 mL allowing pipette rinsing and sampling to take place whilst leaving 40 mL remaining for the growth of the test organism.

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. For the second study the following test concentrations without parallel replicates were prepared 0.02, 0.057, 0.15, 0.39, and 1.0 mg/L.

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 μS/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:

For the study 1: 0.005, 0.013, 0.031, 0.08 and 0.19 mg/L
For the study 2: 0.02, 0.057, 0.15, 0.39 and 1.0 mg/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).

- De-ionized water
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 S/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure.

-Test flasks
The test was performed in sterilized 100 mL Erlenmeyer flasks containing 40 mL of mineral salts in spring water. The test flasks were closed with cotton wool stoppers.

- Culturing cabinet and test conditionsThetest wascarriedoutin atemperature-controlledilluminated orbitalincubatorinwhichthetemperaturewasmaintained at23 ±2°C. UniformIlluminationwas providedin thespectralrange of400to700nm by usingfluorescent lamps atadistance ofabout0.36±0.02m from thealgalcultures.Thelight intensitywasin the rangeof60 to120 µE·m-2·s-1. The test vessels were agitatedcontinuouslyat aspeedsufficienttoprevent sedimentationofthe algae(100rpm approx).

- Other Apparatus
Apparatus was used according to the relevant Standard Operation Procedure as detailed in the study plan. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. The light intensity was measured with a light intensity meter.

- Test concentrations:
A range finding test was conducted using test solutions prepared as described previously. 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 in the first test. Six replicates without the test substance were also prepared as a control. For the second study the following test concentrations without parallel replicates were prepared 0.02, 0.057, 0.15, 0.39, and 1.0 mg/L.

- 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 for both tests.

- Determination of cell concentrations
Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relationship between absorbance and cell density, a calibration curve was made which is checked yearly as part of the GLP laboratory maintenance. From the relation between absorbance (E) and counted cell number per mL (N) the following calibration curve was determined using linear regression:
 
N = 2.0×106´E – 1.81×105  (R2= 0.992)
 
- Determination of pH, temperature, light, and purity of algae
The pH of all samples and controls were measured at the beginning and at the end of the test using a pH meter. The temperature in the culturing apparatus was continuously measured by a temperature sensor and data recorder which was read out at the end of the test. The light intensity was measured at the beginning and at the end of the test using a light intensity meter. At the end of the test five random samples were microscopically checked for purity of the algal culture.

- Quality control of the algae
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the guidelines. The observed EC50 values should be between 0.25 and 2.0 mg/L.


-General test principles and procedures   
Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with natural surface water. The Erlenmeyer flasks were then filled with test medium up to the appropriate volume using a calibrated dispenser pump. Adequate amounts of test substance were then added from the stock solution (see below) to achieve the desired test concentrations in a total volume of 40 mL. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control and a parallel replicate of every concentration without algae were included. The absorbance in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Test medium was used as a blank in the spectrophotometer.

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.:

0.054 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Results from study 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.102 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Results from study 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.058 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Results from study 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.52 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Results from study 2

Details on results:

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

- Definitive test 1
During the definitive study the test organisms displayed less sensitivity to the test substance than observed in the range finding test. Due to this the dose response curve generated was not optimal. All endpoints could be determined and the test met all validity criteria. The study was however repeated by amendment at adjusted concentrations to enable a dose response curve with confidence limits to be generated.

The ErC10 was calculated as 0.112 mg/L x 0.39 = 0.044 mg/L (Growth Rate) and the ErC50 (72h) was calculated as 0.20 mg/L x 0.39 = 0.078 mg/L (Growth rate) [Nominal values corrected for absorbance to glass]

The NOEC was calculated as 0.08 mg/L x 0.39 = 0.031 mg/L and the LOEC
0.19 mg/L x0.39 =0.074 mg/L (Growth rate) [Nominal values corrected for absorbance to glass].

Dose effect and NOEC / LOEC data are given in Figures 5 & 6 as nominal data only. Raw data is reported in Table 2.

Calculation methods were identical as indicated for the second test.

- Definitive test 2
A dose response curve with confidence limits was achieved with the test data and all endpoints could be calculated.

EC10 and EC50 values were 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 results of the absorbance measurements are presented in Table 3. The absorbance values at the highest concentration were artificially set at 100 % inhibition due to negative measurements being made. Growth inhibition based on the area under the growth curve and the specific growth rate, are also displayed here. Calculations were carried out according to Annex 2 and an internally GLP validated spreadsheet.

Table 5 shows the average specific growth rates and corresponding coefficients of variation as required by the guidelines quality criteria. Criteria for variation in the control was met. Variation for section by section growth rates was exceeded. This was due to the closure of the test vessels during the second study. This had however no effect on the endpoints when comparing the results of both tests.

The graphical representations of the test results are given in Figures 3 & 4 and all dose effect and NOEC / LOEC data are given in Figures 1 & 2 as nominal data only.

Inhibition based on growth rate is considered the primary end point for regulatory purposes. Inhibition based on biomass has also been calculated and this is displayed in figure 2 should this be required by other regulatory authorities.

Chemical analysis showed the test substance to be completely stable. For this reason nominal concentrations corrected for absorbance to glass were used to express the endpoints.

The ErC10 was calculated as 0.137 mg/L x 0.39 = 0.054 mg/L (Growth Rate) and the ErC50 (72h) was calculated as 0.264 mg/L x 0.39 = 0.102 mg/L (Growth rate) [Nominal values corrected for adsorption to glass]

The NOEC was calculated as 0.15 mg/L x 0.39 = 0.058 mg/L and the LOEC as
0.39 mg/L x 0.39= 0.152 mg/L. (Growth rate) [Nominal values corrected for absorbance to glass].


- pH, temperature, light and purity of algae
The pH in the prepared test medium in both the first and second studies was slightly higher than the normal range at 8.6 and 8.9 respectively for the first and second studies. The test medium was adjusted to approximately 8 so as to be in the optimal range for the test organism.

The pH measurements in Tables 6&7 show a maximum increase of 0.7 and 2.9 pH units in a control replicate. The temperature varied from 22.1 to 22.3 °C and 21.8 to 22.1ºC during the first and second tests respectively. The light intensity was
101.4 and 102.7 and 101.7 and 101.2 µmol·m-2·s-1 at the beginning and end of the test first and second tests respectively. All measurements are in accordance with the required conditions described in the study plan and in the guideline with the exception of the pH increase observed in the second test due to the closed system. The five random samples checked at the end of the test were not contaminated with excessive levels of bacteria.

The reason for the out of range pH increase was previously mentioned briefly. Whilst reducing the growth in the control (and other replicates) slightly the growth increase was still within acceptable guideline limits. All vessels were tested in identical media and hence under the same conditions. The higher than normal pH increase is therefore not detrimental to the reliability of the toxicity result this is further confirmed by comparison of toxicity data from both tests.

- Chemical Analysis
See tables 1a & 1b for analytical results data for the second test and Annex 4 for the full analytical report.

Analytical quantification in the first study demonstrated a good initial level of the test substance in the stock solution and in the test replicates at the start of the test indicating correct dosing of the test material. As would be expected with strongly adsorbing substances the analytical measurements at the end of the test are very low and heavily influenced by the presence of organic material and by the glass surfaces of the test vessels.

During the second study the additional analytical measurements from extracting the entire test vessel demonstrated almost complete recovery of the test substance indicating that no degradation took place during the study period. However the extractions of the test vessels indicated that a significant amount of the test material was bound to glass. The exposure concentrations for toxicological endpoints were therefore based on nominal concentrations corrected for the average adsorption to glass.

- Average adsorption to glass (see table 1b)
0.034/0.057 X100 =60%
0.091/0.150 X 100 = 61%

61% of the nominal concentration was bound to glass. 39 % of the nominal was therefore used for correction of the nominal ecotoxicological endpoints (see above). Both the first and the second study were corrected in this manner so as to allow direct comparison of results.

QUALITY CRITERIA
The following quality criteria have been met:

• The cell density of the controls increased at least a factor 16 within 72 hours. See Table 5.
• The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7% (See Table 4).
• 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 quality criteria for the analytical method were met.

Analytical quality Criteria (see below table) :

The following criterion was not met (in second test only):
- The mean coefficient of variation for section-by-section specific growth rates in the control did exceed 35 % (See Table 5). This has however no influence on the ecotoxicological endpoints determined. This is evident when comparing calculated values to the first study in which all criteria were met. The determined endpoints differ only marginally from each other.

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. Details of the calculations are given in Annex 3. 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 calculated 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 Dunett’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 TOXCALCtm version V 5.0.23 program and Algal 2A spreadsheet. 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 should these be required and are presented in the tables and figures section.
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.

Analytical quality Criteria (see below):

 

Parameter	Limit	Determined
Squared regression coefficient (R^2)	>= 0.98	>= 0.997
Calibration curve back calculation	80 % or >	81.5 - 104.8
Repeatability (CV (%)) at lowest standard	<= 20	<= 3.6
Repeatability (CV (%)) at Highest standard	<= 20	<=0.6
LOQ (µg/L)	10	<= 0.07
System stability (deviation % from cal. stand.)	<=10	<= 5.3
Recovery from medium (%)	>= 60 and <= 120	95.1 - 96.1

TABLES AND FIGURES

 Table:1a Chemical analysis results first test (corrected for A.I.) of each component

Sample mg/L (Nominal)	0h (mg/L)	72h (mg/L)	72h parallel (mg/L)
control	< LOQ	0.002	--
0.005	0.002	0.003	0.001
0.013	0.008	0.004	0.002
0.031	0.020	0.009	0.006
0.08	0.065	0.021	0.010
0.19	0.147	0.035	0.025
Stock 105 mg/L	98.4

Data not used further

Table:1b Test concentrations second test corrected for active ingredient (A.I.)

sample	Concentration 0h (mg/L) (corrected for A.I.)	Concentration 72h I (mg/L) (corrected for A.I.)	Concentration 72h II (mg/L) (corrected for A.I.)	Concentration 72h III (mg/L) (corrected for A.I.)
control	< LOQ	0.016	0.006
0.02 mg/L	0.015	0.017	0.019
0.057 mg/L	0.046	0.044	0.045	0.034
0.15 mg/L	0.123	0.126	0.126	0.091
0.39 mg/L	0.334	0.366	0.368
1.0 mg/L	0.854	0.966	0.905
Stock	55.9

               I- Extracted with leachingsolution

II-Extracted with acidified leaching solution

III-    Extraction ofglasswith leachingsolution

 Correction for composition: Values reported are corrected for composition. See Annex 4 for all results and an example calculation.

Table 2

Raw data first study

 

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
0.005	0.007	0.038	0.166	0.723	13.152		0.063
0.005	0.007	0.034	0.189	0.760	14.052		0.065
0.005	0.008	0.032	0.171	0.690	12.672		0.062
Mean	0.007	0.035	0.175	0.724	13.292	-7.6	0.063	-1.5
0.013	0.007	0.036	0.177	0.745	13.632		0.065
0.013	0.009	0.032	0.171	0.742	13.236		0.062
0.013	0.008	0.034	0.181	0.736	13.512		0.064
Mean	0.008	0.034	0.176	0.741	13.460	-8.9	0.064	-2.2
0.031	0.008	0.033	0.184	0.791	14.220		0.064
0.031	0.007	0.034	0.184	0.775	14.112		0.065
0.031	0.007	0.022	0.019	0.049	1.152		0.023
Mean	0.007	0.030	0.129	0.538	9.828	20.5	0.059	5.5
0.08	0.008	0.032	0.145	0.649	11.556		0.061
0.08	0.007	0.030	0.128	0.657	11.256		0.063
0.08	0.008	0.031	0.133	0.593	10.572		0.060
Mean	0.008	0.031	0.135	0.633	11.128	9.9	0.061	2.2
0.19	0.008	0.016	0.022	0.090	1.512		0.031
0.19	0.007	0.020	0.033	0.112	2.196		0.036
0.19	0.008	0.018	0.033	0.111	2.076		0.035
Mean	0.008	0.018	0.029	0.104	1.928	84.4	0.034	44.9

 

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

Table 3: Raw data second study

 

Concentration	ABSORBANCE   Time (hours)				AUC	Inhibition %	SGR	Inhibition %
(mg/L)	0	24	48	72
Control	0.013	0.055	0.237	0.403	11.064		0.049
Control	0.012	0.053	0.313	0.475	13.764		0.053
Control	0.011	0.065	0.336	0.493	14.880		0.054
Control	0.013	0.059	0.310	0.447	13.440		0.051
Control	0.013	0.064	0.317	0.435	13.584		0.051
Control	0.014	0.061	0.324	0.445	13.740		0.050
Mean	0.013	0.060	0.306	0.450	13.412		0.051	0.0
0.020	0.014	0.066	0.344	0.470	14.640		0.052
0.020	0.013	0.058	0.325	0.457	13.896		0.053
0.020	0.015	0.059	0.333	0.468	14.124		0.051
Mean	0.014	0.061	0.334	0.465	14.220	-6.0	0.052	-1.4
0.057	0.019	0.059	0.263	0.430	11.748		0.050
0.057	0.019	0.064	0.314	0.421	12.984		0.051
0.057	0.019	0.060	0.305	0.463	13.176		0.052
Mean	0.019	0.061	0.294	0.438	12.636	5.8	0.051	0.8
0.150	0.018	0.042	0.189	0.355	8.724		0.050
0.150	0.019	0.048	0.151	0.247	6.600		0.042
0.150	0.021	0.032	0.130	0.242	5.532		0.042
Mean	0.019	0.041	0.157	0.281	6.952	48.2	0.045	13.2
0.390	0.021	0.020	0.037	0.059	0.816		0.021
0.390	0.020	0.020	0.033	0.036	0.504		0.014
0.390	0.021	0.014	0.030	0.013	-0.048		-0.007
Mean	0.021	0.018	0.033	0.036	0.424	96.8	0.014	73.2
1.000	0.032	0.011	0.014	0.0004	-1.315		Error
1.000	0.022	0.011	0.017	-0.0009	-0.659		Error
1.000	0.033	0.022	0.024	0.008	-0.780		Error
Mean	0.029	0.015	0.018	0.003	-0.918	>100.0		>100.0

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

Note: For calculation purposes data from the highest concentration was set as a maximum of 100 % inhibition

Table 4: Average specific growth rates and coefficients of variation during the whole test period in the control and section by section(Validity Criteria) in the first study.

 

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

Table 5: Average specific growth rates and coefficients of variation during the whole test period in the control and section by section (Validity Criteria) in the second study.

 

0-24	24-48	48-72	72h test mean	stdev	%CV	Mean %CV
0.060	0.061	0.022	0.0477	0.02215	46.44	58.67
0.062	0.074	0.017	0.0511	0.02981	58.36
0.074	0.068	0.016	0.0528	0.03203	60.64
0.063	0.069	0.015	0.0491	0.02950	60.05
0.066	0.067	0.013	0.0488	0.03081	63.18
0.061	0.070	0.013	0.0480	0.03044	63.35
		mean	0.0496
		stdev	0.0020
		CV%	3.9863

Table 6: Measurement of pH (first test)

Nominal test concentration (mg/L)	Time (hours)
	0	72
I-ControlII-ControlIII-ControlIV-ControlV-Control VI- Control	8.2	8.9
	8.2	9.4
	8.2	9.5
	8.2	9.5
	8.2	9.1
	8.2	9.0
I 0.005 II0.005 III0.005	8.2	9.5
	8.3	9.5
	8.3	9.3
I0.013 II0.013 III0.013	8.3	9.3
	8.3	9.4
	8.3	9.4
I 0.031 II0.031 III0.031	8.3	9.4
	8.3	9.4
	8.3	8.6
I0.08 II   0.08 III   0.08	8.3	9.2
	8.3	9.1
	8.3	9.1
I0.19 II   0.19 III   0.19	8.3	8.6
	8.3	8.6
	8.3	8.5

 

Table 7: Measurement of pH (Second test)

 

Nominal test concentration (mg/L)	Time (hours)
	0	72
I-ControlII-ControlIII-ControlIV-ControlV-Control VI- Control	8.2	11.1
	8.2	11.3
	8.2	11.3
	8.2	11.3
	8.2	11.2
	8.2	11.2
I 0.005 II0.005 III0.005	8.2	11.2
	8.2	11.2
	8.2	11.1.
I0.013 II0.013 III0.013	8.2	10.9
	8.2	10.9
	8.2	11.0
I 0.031 II0.031 III0.031	8.2	10.5
	8.2	10.1
	8.2	9.9
I0.08 II   0.08 III   0.08	8.2	8.7
	8.2	8.3
	8.2	8.3
I0.19 II   0.19 III   0.19	8.2	8.1
	8.2	8.1
	8.2	8.0

For figures 1 -6 Annex 3, Annex 4 and CoA, please refer to the attachments.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC10 and ErC50 of test substance for Pseudokirchnerella subcapitata was 0.054 and 0.102 mg a.i./L, respectively. The NOEC and LOEC for effect on algal growth rate was determined to be 0.058 and 0.152 mg a.i./L respectively

Executive summary:

A study was conducted to determine the toxicity of the test substance, C16-18 ADBAC (purity: 98.2%), on the exponential growth of the algae Pseudokirchnerella subcapitata according to OECD Guideline 202 and EU Method C.3, in compliance with GLP. Algae were exposed to nominal concentrations of 0.005, 0.013, 0.031, 0.08 and 0.19 mg a.i./L in the first study and 0.02, 0.057, 0.15, 0.39 and 1.0 mg a.i./L in the second study. Due to the absence of a full concentration-response curve in the first study, the test was repeated with an optimised test concentration range to allow the calculation of a more robust endpoint for risk assessment purposes. The data from the second test allowed a concentration-response curve with confidence limits to be generated. Therefore, only results of second study has been presented here. Algae cell concentrations were determined spectrophotometrically. Chemical analysis performed at the beginning, middle and the end of the test using LC-MS/MS showed the test substance to be completely stable. Due to this reason, the nominal concentrations corrected for absorbance to glass were used to express the endpoints. All the validity criteria were fulfilled, except that the mean coefficient of variation for section-by-section specific growth rates in the control exceeded 35%. This was due to the closure of the test vessels during the study and was however considered to have no effect on the endpoints when comparing the results of both tests. Under the study conditions, the 72 h ErC10 and ErC50 of test substance for Pseudokirchnerella subcapitata was 0.054 and 0.102 mg a.i./L, respectively. The NOEC and LOEC for effect on algal growth rate was determined to be 0.058 and 0.152 mg a.i./L respectively (Kean, 2012).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From 05 March, 2004 to 19 March, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not specified

Test organisms (species):

Skeletonema costatum

Details on test organisms:

The strain NIVA BAC 1 was used.
Algal medium 1/2 'T' from Guillard and Ryther (1962) was used.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

20± 2.4°C

pH:

8.51 to 8.64

Nominal and measured concentrations:

0.1, 0.5, 2.5 and 10 mg/L (nominal whole product)

.

Details on test conditions:

Test method:
- Growth inhibition test (72h) with the marine diatom Skeletonema costatum was performed
- Initially, a preliminary (range finding) test with two parallel test concentrations, followed by a definitive test with 3 parallel concentrations and 6 controls was performed.
- Inhibition was measured as a reduction in growth rate, (the growth rate is the increase in cell density per unit time) relative to control cultures grown under identical conditions.
The EC50-, EC90- , EC10 - and NOEC- values were determined. The EC50-value is defined as the dosage of the test substance that gives a 50% reduction in growth rate relative to control. The NOEC-value is defined as the highest dosage of the test substance that gives no observed effect on the growth rate. The cell density was measured by fluorescence (Turner 10-100 R fluorometer).
The average specific growth rate (mav) is calculated based on this equation:
mav =In (Xt)- In (Xo)/t
Xt= final algal density
Xo= initial algal density
T= test

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to 0.207 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to 0.096 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

ca. 0.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to 0.34 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to 0.032 mg a.i./L

Details on results:

In this test, all the control parameters were within the recommended ranges. According to the EEC classification system (EEC, 1993), a test substance is classified as very toxic if EC50 < 1 mg/L, as toxic if EC50 = 1-10 mg/L, and as harmful if EC50 >10-100 mglL based on algae. Based on this, the test substance PR-4951 can be classified as very toxic according to the EEC classification system

Results with reference substance (positive control):

One test concentration (1.5 mg/L) of the reference substance, 3,5-dichlorphenol, was analyzed. The growth rate inhibition observed was 40% after 72h.

Reported statistics and error estimates:

Method for data handling: The mean values of EC50, EC90, EC10, NOEC and the confidence limits are calculated from the computer program Excel. The dosage-response curve is printed out, and the EC-values are estimated by drawing lines by hand

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50 and ErC10 (Skeletonema costatum) of the read across substance were 0.26 and 0.12 mg/L, respectively (i.e., equivalent to 0.207 mg a.i./L and 0.096 mg a.i./L). The 72h NOEC was 0.04 mg/L which corresponds to 0.032 mg a.i./L.

Executive summary:

A study was conducted to determine the toxicity to marine algae of the read across substance, C12 -14 ADBAC (60-100% active), according to ISO 10253, in compliance with GLP. Skeletonema costatum were exposed for 72 h at nominal concentrations of 0, 0.1, 0.5, 2.5 and 10 mg/L, i.e., equivalent to 0, 0.08, 0.4, 2 and 8 mg a.i./L considering 80% as mean active. Analytical dose verification was not performed. The cell density was measured by fluorescence. To ensure reproducibility, a single concentration 1.5 mg/L of reference substance 3,5-dichlorophenol was included in the study, which indicated 40% mortality after 72 h. All validity criteria were fulfilled. Under the study conditions, the 72 h ErC50 and ErC10 (Skeletonema costatum) of the read across substance were 0.26 and 0.12 mg/L, respectively (i.e., equivalent to 0.207 mg a.i./L and 0.096 mg a.i./L). The 72 h NOEC was 0.04 mg/L which corresponds to 0.032 mg a.i./L (Sundfor, 2004). Based on the results of the read across study, similar effect levels can be expected for the test substance.

Description of key information

The freshwater 72 h ErC10 and ErC50 values of the test substance for effects on growth rate was determined to be 0.054 and 0.102 mg a.i./L (nominal and corrected for absorbance to glass). Further, based on the results of a read across study with C12-14 ADBAC, the marine water 72 h ErC50 and ErC10 values of the test substance for effects on growth rate is considered to be 0.207 and 0.096 mg a.i./L (nominal) respectively. These respective ErC10 and ErC50 values in fresh and marine water have been considered further for hazard/risk assessment.  

Key value for chemical safety assessment

EC50 for freshwater algae:

102 µg/L

EC50 for marine water algae:

207 µg/L

EC10 or NOEC for freshwater algae:

54 µg/L

EC10 or NOEC for marine water algae:

96 µg/L

Additional information

Freshwater:

A study was conducted to determine the toxicity of the test substance, C16-18 ADBAC (purity: 98.2%), on the exponential growth of the algaePseudokirchnerella subcapitataaccording to OECD Guideline 202 and EU Method C.3, in compliance with GLP. Algae were exposed to nominal concentrations of 0.005, 0.013, 0.031, 0.08 and 0.19 mg a.i./L in the first study and 0.02, 0.057, 0.15, 0.39 and 1.0 mg a.i./L in the second study. Due to the absence of a full concentration-response curve in the first study, the test was repeated with an optimised test concentration range to allow the calculation of a more robust endpoint for risk assessment purposes. The data from the second test allowed a concentration-response curve with confidence limits to be generated. Therefore, only results of second study has been presented here. Algae cell concentrations were determined spectrophotometrically. Chemical analysis performed at the beginning, middle and the end of the test using LC-MS/MS showed the test substance to be completely stable. Due to this reason, the nominal concentrations corrected for absorbance to glass were used to express the endpoints. All the validity criteria were fulfilled, except that the mean coefficient of variation for section-by-section specific growth rates in the control exceeded 35%. This was due to the closure of the test vessels during the study and was however considered to have no effect on the endpoints when comparing the results of both tests. Under the study conditions, the 72 h ErC10 and ErC50 of test substance forPseudokirchnerella subcapitatawas 0.054 and 0.102 mg a.i./L, respectively. The NOEC and LOEC for effect on algal growth rate was determined to be 0.058 and 0.152 mg a.i./L respectively (nominal corrected for absorbance to glass) (Kean, 2012). 

Marine water

A study was conducted to determine the toxicity to marine algae of the read across substance, C12-14 ADBAC (60-100% active), according to ISO 10253, in compliance with GLP.Skeletonema costatumwere exposed for 72 h at nominal concentrations of 0, 0.1, 0.5, 2.5 and 10 mg/L, i.e., equivalent to 0, 0.08, 0.4, 2 and 8 mg a.i./L considering 80% as mean active. Analytical dose verification was not performed. The cell density was measured by fluorescence. To ensure reproducibility, a single concentration 1.5 mg/L of reference substance 3,5-dichlorophenol was included in the study, which indicated 40% mortality after 72 h. All validity criteria were fulfilled. Under the study conditions, the 72 h ErC50 and ErC10 (Skeletonema costatum) of the read across substance were 0.26 and 0.12 mg/L, respectively (i.e., equivalent to 0.207 mg a.i./L and 0.096 mg a.i./L). The 72 h NOEC was 0.04 mg/L which corresponds to 0.032 mg a.i./L (Sundfor, 2004).  

Based on the available information, the freshwater 72 h ErC10 and ErC50 values of the test substance for effects on growth rate was determined to be 0.054 and 0.102 mg a.i./L (nominal and corrected for absorbance to glass). Further, based on the results of a read across study with C12-14 ADBAC, the marine water 72 h ErC50 and ErC10 values of the test substance for effects on growth rate is considered to be 0.207 and 0.096 mg a.i./L (nominal) respectively. These respective ErC10 and ErC50 values in fresh and marine water have been considered further for hazard/risk assessment.