Protein hydrolyzates, wool, reaction products with 3-chloro-2-hydroxypropyl-stearyl-dimethylammonium 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 04 September 2018 to 07 September 2018

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:

yes

Remarks:

in the light intensity and temperature range; however in the opinion of the study director, this deviation had no effect on the validity and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analysis of the samples for the verification of exposure concentrations was performed using HPLC.

Details on sampling:

Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.

Vehicle:

yes

Remarks:

Sterilised deionised water with added nutrients

Details on test solutions:

Information provided by the Sponsor indicated that the sample was completely soluble in water. The test concentrations for both the range-finding test and definitive test were prepared separately by the addition of the test sample to nutrient growth medium in volumetric flasks of adequate volume to provide enough solution for testing and subsequent water quality measurements.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata strain: CCAP 278/4 received 15 May 2018.
Source: Culture Collection of Algae and Protozoa, SAMS Ltd, Scottish Marine Institute, OBAN, Argyll PA37 1QA, Scotland, United Kingdom

Culture conditions:
Temperature: 21 - 21.3 deg C.
Illumination: 5480 - 7910 lux continuous white light.
Orbiting: set to 200 rpm
Culture media: Deionised water with added nutrients according to OECD 201.

The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium (OECD 201) which was prepared by adding appropriate amounts of nutrient stocks to deionised water (sterilised by autoclaving at 120°C for 15 minutes) at a pH of 7.51 (adjusted to: 8.15).

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.4 – 21.4°C

pH:

7.78 – 8.40

Nominal and measured concentrations:

Nominal: 0 (control), 0.010, 0.032, 0.10, 0.32, 1 and 3.2 mg/L
Measured: 0, -0.347, -0.314, 0.083, 0.290, 0.958 and 2.976 mg/L
The results indicate that the measured concentrations for 0.10, 0.32, 1 and 3.2 mg/L nominal concentrations remained within 82.961 – 95.838% of nominal concentrations. The 72 h EC50 was at a concentration at which the analytical recoveries were >90% of nominal concentrations . Therefore effect concentrations are reported as nominal concentrations of test substance as received.

Details on test conditions:

Test methods and conditions

The test was carried out according to the procedures given below, based on the guidelines produced by OECD 201: Alga Growth Inhibition Test.

Chemex SOP reference: E203 “Algal Growth Inhibition Test (Freshwater)”
Preliminary test method: A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100 mg/L. The duration of the preliminary study was 72 ± 2 h. There was a single replicate at each concentration.
Preliminary test results: Data from the preliminary test identified the 72-h EC50 as being between 1 and 10 mg/L (by growth rate).

Nominal concentration (mg/L) Percent reduction in growth rate
0 - 72 h
0.1 3
1.0 28
10 100
100 100

All concentrations of the test substance are reported as nominal as received.

Definitive test method:
Test period: 04 to 07 September 2018
Test duration: 72 ± 2 h
Test volume: 100 mL
Test vessel: 250 mL conical flask
Number of replicates: Six control flasks, three replicates at each test concentration.

Test concentrations:
Nominal: 0 (control), 0.010, 0.032, 0.10, 0.32, 1 and 3.2 mg/L

Composition of medium: Sterilised deionised water with added nutrients according to the OECD 201 standard.
Algal Test inoculum: From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.
Initial pH at 0 H: 8.15 (Required: 8.1±0.1)
pH range in control and test concentrations throughout test: 7.78 – 8.40
Temperature range within incubator throughout test: 20.4 – 21.4°C (Required: 21 - 24 ± 2°C)
Illumination range within incubator throughout test: 5480 - 8180 Lux (Required: 6x103-8x103 Lux ± 15% of recorded mean)
Orbital shaking: 200 rpm (Required: 200-250rpm)

Water quality measurements:
The temperature (to 0.1 deg C) and light intensity (lux) within the incubator was recorded at the beginning of the study, after 24, 48 h and at the end of the 72 h test period. The pH (to 0.01) and temperature (to 0.1 deg C) were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-h test period.

Observations/frequency:
Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 h for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 h (±2h) for the test concentrations. The cell counts were made using a haemocytometer and microscope.

Analysis of test substance:
Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

Calculation of results:
Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996(2).

Reference Substance:
A separate reference study (ENV 11396) was run from 16 to 19 January 2017 using potassium dichromate (K2Cr2O7) as a means of checking the test procedure and sensitivity of the test species. ISO 8692:2012 quotes the 72-h ErC50 as 1.19mg/L (SD = 0.27), the result obtained in the reference test should be in the range 0.65 and 1.73 mg/L (mean value ± 2 x standard deviation). The ErC50 obtained was 1.06 mg/L and all validity criteria were met.

Study plan deviation
Definitive test: states that the light should be at an “intensity in the range 6000 - 8000 lux at the level of the test flasks and will not vary by more than ± 15% from the mean value across all test vessels”. A minimum lux value of 5480 and a maximum of 8420 lux was recorded, 520 lux below and 420 lux above the recommended range. The range was also outside the ± 15% recommended range stated in OECD 201. The study plan also states that the culture should be within this range, but a minimum value of 5480 lux was recorded. Also, study plan section 9.4 Additional Study Details: Definitive test: also states the temperature range within the incubator throughout test should be 21-24°C. A minimum temperature of 20.4°C was recorded, 0.6°C below the OECD 201 recommended range. The cell density in the control cultures increased by the required amount (a factor of at least 16) within the 72-h test period. Also, there were no cell morphological abnormalities within the control units. Therefore, any changes in cell density in the test concentrations can be attributed to the test substance. In the opinion of the study director, this had no detrimental effect on the validity and integrity of this study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

ca. 0.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.53-1.16

Remarks:

determined by Maximum Likelihood-Logit; (i.e., equivalent to 0.15 mg a.i./L)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Determined by Bonferroni t Test

Remarks:

(i.e., equivalent to 0.026 mg a.i./L)

Details on results:

The control cell density should increase by a factor of more than 16 in 72 h, corresponding to a specific growth rate of 0.92 d-1. The measured control cell density increase was recorded as 77.2, corresponding to a specific growth rate of 1.446 d-1. The variation coefficient of the control specific growth rate should not exceed 7% for the 72 h period and 35% for each time period. The variation coefficient for the 72h period was 3.43% and for each time period 19.08%. These fulfil the validity criteria of the study and it is therefore compliant with the OECD Guideline for Testing of Chemicals reference 201 Alga, Growth Inhibition Test 2011.

Results

Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 h for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 h (±2h) for the test concentrations. All results in this study are calculated from the measured cell densities.

 

Cell density measurements

Mean initial cell density: Approximately 1 x 10⁴cells/ml based upon inoculation volume, not counted microscopically.

Nominal concentration (mg/L)	Replicate	Cell density measurements (cells/ml x 10^4)
		24 h	48 h	72 h
0 (Control)	1	5.5	28.0	93.7
	2	4.3	20.7	74.3
	3	3.3	17.0	64.7
	4	2.3	18.3	66.0
	5	3.8	19.7	76.0
	6	4.7	24.7	88.7
	Mean	4.0	21.4	77.2
0.010	1	4.7	27.3	96.7
	2	3.3	19.3	70.0
	3	5.7	26.0	69.0
	Mean	4.6	24.2	78.6
0.032	1	6.3	17.0	65.7
	2	4.3	18.0	63.7
	3	5.0	29.0	68.3
	Mean	5.2	21.3	65.9
0.10	1	3.7	19.0	52.7
	2	3.7	17.7	53.0
	3	5.0	21.0	55.3
	Mean	4.1	19.2	53.7
0.32	1	3.0	13.3	37.3
	2	3.7	17.7	43.0
	3	3.0	15.3	38.3
	Mean	3.2	15.4	39.5
1	1	1.3	2.3	1.7
	2	0.7	3.3	1.3
	3	1.3	3.7	3.7
	Mean	1.1	3.1	2.2
3.2	1	3.3	8.7	2.0
	2	1.0	4.0	1.0
	3	1.7	8.0	5.0
	Mean	2.0	6.9	2.7

 

Growth curves of logarithm of cell density against time are given in Graph 1 (Kindly refer to the attached background material section of the IUCLID).

 

Test observations

The algal cells were examined microscopically during the determination of the cell density, all cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed. When observed visually, the control, 0.010. 0.032, and 0.10 mg/L conical flasks appeared clear and colourless after 24 h and green after 48 and 72 h. The 0.32 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 1 and 3.2 mg/L conical flasks remained clear and colourless throughout the test.

 

Percent inhibition

Nominal concentration (mg/L)	Percent reduction in growth rate
	0 - 48 h	0 - 72 h
0.010	-4	0
0.032	1	3
0.10	3	8
0.32	10	15
1	64	84
3.2	38	82

Note: Negative numbers indicate an increase in growth compared to the unexposed controls.

 

EC_x, NOEC values by yield (E_yC_x) and growth rate (E_rC_x)

 

Exposure Period (h)	ErCx value mg/L (95% confidence limits)
	ErC10	ErC20	ErC50
0 to 48	0.09 (0.004 – 0.25)	0.31 (0.05 – 0.63)	2.51 (1.30 – 10.22)
0 to 72	0.25 (0.17 – 0.32)	0.34 (0.26 – 0.41)	0.58 (0.49 – 0.68)
NOEC (0-72h)	0.1 mg/L (Determined by Bonferroni t Test)^$
Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit. ^$Normality of distribution and equality of variances could not be confirmed. All concentrations of the test substance are reported as nominal as received.

 

Test validity criteria

The control cell density should increase by a factor of more than 16 in 72 h, corresponding to a specific growth rate of 0.92 d^-1. The measured control cell density increase was recorded as 77.2, corresponding to a specific growth rate of 1.446 d^-1. The variation coefficient of the control specific growth rate should not exceed7% for the 72h period and 35% for each time period. The variation coefficient for the 72h period was 3.43% and for each time period 19.08%. These fulfil the validity criteria of the study and it is therefore compliant with the OECD Guideline for Testing of Chemicals reference 201 Alga, Growth Inhibition Test 2011.

Control growth rate data coefficient of variation calculations 

Control replicate no.	Cell density (1.0 x 10^4cells/ml)				0-72 h average specific growth rates
	0 h	24 h	48 h	72 h
1	1.0	5.5	28.0	93.7	1.51
2	1.0	4.3	20.7	74.3	1.44
3	1.0	3.3	17.0	64.7	1.39
4	1.0	2.3	18.3	66.0	1.40
5	1.0	3.8	19.7	76.0	1.44
6	1.0	4.7	24.7	88.7	1.50
			Mean:		1.45
			Standard deviation:		0.0497
			Coefficient of variation:		3.43

Note: 0-h cell density based upon inoculation volume and not counted microscopically.

 

Control replicate no.	Day-by-day specific growth rates			Coefficient of variation
	0-24 h	24-48 h	48-72 h
1	1.70	1.63	1.21	17.51
2	1.46	1.57	1.28	10.19
3	1.19	1.64	1.34	16.48
4	0.83	2.07	1.28	45.05
5	1.34	1.65	1.35	12.18
6	1.55	1.66	1.28	13.06
Mean coefficient of variation:				19.08

Note:0-h cell density based upon inoculation volume, not counted microscopically.

 

Discussion

The definitive test conducted from 04 to 07 September 2018 was performed according to the OECD 201 (2011) guideline. The growth curves illustrated in Graph 1 demonstrate that the algae in the control were in logarithmic growth for the duration of the study. The 48 h EC(r)₅₀of test substance to Pseudokirchneriella subcapitata was 2.51 mg/L (determined by Maximum Likelihood-Logit). Graphical representations of the 0-48 h EC(r)_xvalue is given in Graph 2. The 72 h EC(r)₅₀of test substance to Pseudokirchneriella subcapitata were 0.58 mg/L (determined by Maximum Likelihood-Logit). Graphical representations of the 0-72 h EC(r)_xvalue is given in Graph 4. The 0 to 72-h NOEC(r) was 0.1 mg/L (determined by Bonferroni t Test) and the 0-72 h LOEC was 0.32 mg/L (determined by Bonferroni t Test). The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test concentrations appeared normal, no abnormalities were observed. All validity criteria for the definitive test were met. The water quality measurements and incubation conditions were within accepted limits. Results for the analytical confirmation of exposure concentrations indicate that the measured concentrations for 0.10, 0.32, 1 and 3.2 mg/L nominal concentrations remained within 82.961 – 95.838% of nominal concentrations. The 72 h EC₅₀ was at a concentration at which the analytical recoveries were >90% of nominal concentrations. Therefore effect concentrations are reported as nominal concentrations of test substance as received. Additional definitive tests were run on 12 to 15 March 2018, 08 to 11 May 2018 and 06 to 09 August 2018 but were not reported due to receipt of new sample with different batch number.

For graphs, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50, ErC10 and NOEC values for the test substance with freshwater green algae, were determined to be 0.58, 0.25 and 0.1 mg/L (nominal) respectively (i.e., equivalent to 0.15, 0.065 and 0.026 mg a.i./L respectively).

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool' (active: 26.1%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100 mg/L. Data from the preliminary test identified the 72-h EC50 1 and 10 mg/L (by growth rate). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 0.010, 0.032, 0.10, 0.32, 1 and 3.2 mg/L in sterilised deionised water (with added nutrients) for 72 h. Inoculum level was adjusted to give an initial cell density of 1 × 104 cells/mL. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 72 h exposure period using HPLC. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h. The results indicated that the measured concentration of the stock solution remained within 82.961 – 95.838% of the nominal concentration. Therefore, effect concentrations were reported as the nominal as received. Cell densities were measured microscopically by direct cell counts on each control replicate at 24 h and on each test replicate at 24, 48 and 72 h (±2 h) using haemocytometer and microscope. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0. All cells within the control and all test concentrations appeared normal; no morphological abnormalities were observed following microscopical observations. When observed visually, the control, 0.010. 0.032, and 0.10 mg/L conical flasks appeared clear and colourless after 24 h and green after 48 and 72 h. The 0.32 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 1 and 3.2 mg/L conical flasks remained clear and colourless throughout the test. The ErC50 and ErC10 values (for growth rate) were calculated to be 0.58 (95% confidence interval: 0.49 - 0.68) and 0.25 (95% confidence interval: 0.17 -0.32) (nominal), respectively. The NOEC value was determined to be 0.1 mg/L (nominal) for growth rate. The NOEC for yield fell below the limit of detection of the analytical method, therefore could not be determined. The measured control cell density increase was recorded as 77.2, corresponding to a specific growth rate of 1.446 per day (which is >0.92 per day). The variation coefficient for the 72 h period was 3.43% (which is below the 7% limit) and for each time period 19.08% (which is below the 35% limit). Therefore, all validity criteria were fulfilled. Under the study conditions, the 72 h ErC50, ErC10 and NOEC values for the test substance with freshwater green algae, were determined to be 0.58, 0.25 and 0.1 mg/L (nominal) respectively (i.e., equivalent to 0.15, 0.065 and 0.026 mg a.i./L respectively) (Chemex, 2018).

Description of key information

Based on results of the study, the 72 h ErC50, ErC10 and NOEC values for the test substance with freshwater green algae, can be considered to be 0.15, 0.065 and 0.026 mg a.i./L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.15 mg/L

EC10 or NOEC for freshwater algae:

0.065 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool' (active: 26.1%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100 mg/L. Data from the preliminary test identified the 72-h EC50 1 and 10 mg/L (by growth rate). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 0.010, 0.032, 0.10, 0.32, 1 and 3.2 mg/L in sterilised deionised water (with added nutrients) for 72 h. Inoculum level was adjusted to give an initial cell density of 1 × 104 cells/mL. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 72 h exposure period using HPLC. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h. The results indicated that the measured concentration of the stock solution remained within 82.961 – 95.838% of the nominal concentration. Therefore, effect concentrations were reported as the nominal as received. Cell densities were measured microscopically by direct cell counts on each control replicate at 24 h and on each test replicate at 24, 48 and 72 h (±2 h) using haemocytometer and microscope. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0. All cells within the control and all test concentrations appeared normal; no morphological abnormalities were observed following microscopical observations. When observed visually, the control, 0.010. 0.032, and 0.10 mg/L conical flasks appeared clear and colourless after 24 h and green after 48 and 72 h. The 0.32 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 1 and 3.2 mg/L conical flasks remained clear and colourless throughout the test. The ErC50 and ErC10 values (for growth rate) were calculated to be 0.58 (95% confidence interval: 0.49 - 0.68) and 0.25 (95% confidence interval: 0.17 -0.32) (nominal), respectively. The NOEC value was determined to be 0.1 mg/L (nominal) for growth rate. The NOEC for yield fell below the limit of detection of the analytical method, therefore could not be determined. The measured control cell density increase was recorded as 77.2, corresponding to a specific growth rate of 1.446 per day (which is >0.92 per day). The variation coefficient for the 72 h period was 3.43% (which is below the 7% limit) and for each time period 19.08% (which is below the 35% limit). Therefore, all validity criteria were fulfilled. Under the study conditions, the 72 h ErC50, ErC10 and NOEC values for the test substance with freshwater green algae, were determined to be 0.58, 0.25 and 0.1 mg/L (nominal) respectively (i.e., equivalent to 0.15, 0.065 and 0.026 mg a.i./L respectively) (Chemex, 2018).