Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-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 18 September 2018 to 21 September 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

in 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: 20.2 - 21.3 deg C.
Illumination: 6040 - 8490 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 6.92 (adjusted to: 8.12).

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.8 – 21.8°C

pH:

7.50 - 8.19

Nominal and measured concentrations:

Nominal: 0 (control), 0.0625, 0.125, 0.25, 0.5, and 1 mg/L

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 0.1 and 1 mg/L (by growth rate) and 0.1 and 1 mg/L (by yield).

Nominal concentration (mg/L) Percent reduction in growth rate Percent reduction in yield
0 - 72 hours 0 - 72 hours
0.1 1 3
1.0 100 100
10 100 100
100 100 100

All concentrations of the test substance are reported as nominal as received and are expressed as loading rates, following preparation of water accommodated fractions (WAF’s).

Definitive test method:
Test period: 18 to 21 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), 0.0625, 0.125, 0.25, 0.5, and 1 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, under conditions described in 2.2 above. Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.
Initial pH at 0 Hours: 8.19 (Required: 8.1±0.1)
pH range in control and test concentrations throughout test: 7.50 - 8.19
Temperature range within incubator throughout test: 20.8 – 21.8°C (Required: 21-24±2°C)
Illumination range within incubator throughout test: 5890 - 7590 Lux (Required: 6x103-8x103 Lux ± 15% of recorded mean)
Orbital shaking: 200rpm (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 hours 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 1.73mg/L (mean value ± 2 x standard deviation). The ErC50 obtained was 1.06mg/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.” It also states that the incubation temperature should be maintained at 21-24 deg C. A minimum lux value of 5890 Lux was recorded, 110 lux below the recommended minimum and temperatures were maintained between 20.8-21.8 deg C. Lux levels also exceeded the ± 15% recommended variation limit. However, these values do not represent a deviation from the OECD 201 guideline. Cell densities in the control cultures increased by the required amount (a factor of at least 16) within the 72-h test period. Therefore, test sample concentration is considered to be the controlling factor in cell density changes in the test treatments. No morphological abnormalities were evident in any of the control vessels. Study plan states that algal pre-cultures should also be maintained within a temperature range of 21-24 deg C and lux range of 6000-8000 lux. However, a temperature range of 20.2-21.3 deg C and lux range of 6040-8490 were recorded.
As pre-culture cell density increased to an acceptable level of 154x104 cells/mL (1x104 required) and no cellular abnormalities were noted, cultures were deemed healthy and fit for testing.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

ca. 0.72 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.53-1.16

Remarks:

(i.e., equivalent to 0.367 mg a.i./L)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

other: growth rate and biomass

Remarks on result:

other: Determined by Bonferroni t Test

Remarks:

(i.e., equivalent to 0.064 mg a.i./L)

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 0.22-0.32

Remarks:

(i.e., equivalent to 0.14 mg a.i./L)

Details on 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.

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 (±2 h) 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 10⁴ cells/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	6.2	20.3	80.7
	2	2.5	15.7	66.3
	3	1.8	13.3	60.3
	4	1.5	13.3	67.0
	5	2.0	13.0	61.7
	6	4.2	13.0	70.3
	Mean	3.0	14.8	67.7
0.0625	1	5.3	19.3	77.0
	2	2.0	12.3	60.3
	3	3.7	17.0	66.7
	Mean	3.7	16.2	68.0
0.125	1	2.0	16.3	73.0
	2	2.0	12.7	55.0
	3	1.7	16.7	61.3
	Mean	1.9	15.2	63.1
0.25	1	2.0	13.7	37.0
	2	1.7	12.3	40.3
	3	2.7	14.0	30.0
	Mean	2.1	13.3	35.8
0.5	1	1.7	13.7	10.3
	2	1.3	20.7	17.0
	3	3.0	12.3	9.3
	Mean	2.0	15.6	12.2
1	1	6.0	12.3	5.7
	2	2.3	6.0	5.3
	3	4.3	5.7	6.0
	Mean	4.2	8.0	5.7

 Growth curves of logarithm of cell density against time are given in Graph 1 (Kindly refer the attached background material section of 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, 0.0625, 0.125 and 0.25 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 0.5 mg/L conical flasks appeared slightly green after 48 and 72 h and 1 mg/L conical flasks appeared clear and colourless thoughout the test.

 

Percent inhibition 

Nominal concentration (mg/L)	Percent reduction in growth rate		Percent reduction in yield
	0 - 48 h	0 - 72 h	0 - 48 h	0 - 72 h
0.0625	-3	0	-10	0
0.125	-1	2	-3	7
0.25	3	15	10	48
0.5	-2	42	-6	83
1	25	59	49	93

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)			EyCxvalue mg/L (95% confidence limits)
	ErC10	ErC20	ErC50	EyC10	EyC20	EyC50
0 to 48	0.85* (0.07-0.16)	0.96* (0.11-0.20)	1.18*  (0.22-0.32)	0.83^*	0.89^*	1.00^*
0 to 72	0.17 (0.04-0.28)	0.30 (0.12-0.42)	0.72 (0.53-1.16)	0.12 (0.07-0.16)	0.16 (0.11-0.20)	0.27 (0.22-0.32)
NOEC (0-72h)	0.125 mg/L (Determined by 72Y Hypothesis test)^$			0.125 mg/L (Determined by Bonferroni t Test)^$
Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit *Confidence limits not possible to determine. ^$Following Shapiro-Wilk’s Test for normality of distribution and Bartlett’s Test which indicated equal variances. 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 67.7, corresponding to a specific growth rate of 1.404 d^-1. The variation coefficient of the control specific growth rate should not exceed 7% for the 72h period and 35% for each time period. The variation coefficient for the 72h period was 2.42%, however the period-by-period variation was 39.36%. As mean cell growth was sustained at an acceptable rate over the course of this study, and as clear EC values could be determined. It is the opinion of the study director that the control variation was not sufficient to prevent an acceptable, accurate and valid assessment of the aquatic toxicity of test substance to Pseudokirchneriella subcapitata under the conditions of this test.

 

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	6.2	20.3	80.7	1.46
2	1.0	2.5	15.7	66.3	1.40
3	1.0	1.8	13.3	60.3	1.37
4	1.0	1.5	13.3	67.0	1.40
5	1.0	2.0	13.0	61.7	1.37
6	1.0	4.2	13.0	70.3	1.42
			Mean:		1.40
			Standard deviation:		0.0339
			Coefficient of variation:		2.42

 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	1.82	1.19	1.38	22.08
2	0.92	1.84	1.44	32.95
3	0.59	2.00	1.51	52.38
4	0.41	2.18	1.62	64.47
5	0.69	1.87	1.56	44.54
6	1.44	1.13	1.69	19.76
Mean coefficient of variation:				39.36

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

 

Discussion

The definitive test reported here was conducted from the 18^th- 21^st September 2018 and was performed in accordance with the OECD 201 (2011) guideline. The growth curves illustrated in Graph 1 demonstrate that the control algae achieved logarithmic growth for the duration of the study. The 48 h E_rC₅₀and E_yC₅₀of test substance to Pseudokirchneriella subcapitata were 1.18 mg/L and 1.00 mg/L respectively (determined by Maximum Likelihood-Logit). Graphical representations of the 0-48 hour E_rC_xand E_yC_xvalues are given in Graphs 2 and 3, respectively. The 72 h E_rC₅₀ and E_yC₅₀ of test substance to Pseudokirchneriella subcapitata were 0.72 mg/L and 0.27 mg/L respectively (determined by Maximum Likelihood-Logit). Graphical representations of the 0-72 h E_rC_xand E_yC_xvalues are given in Graphs 4 and 6 respectively.The 0 to 72-h NOEC(r) was 0.125 mg/L (determined by Bonferroni t Test) and the 0-72 h LOEC was 0.25 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. The water quality measurements and incubation conditions were within accepted limits. The OECD 201 guideline states that the period-by-period coefficient of variation should not exceed 35%, 39.36% was recorded in this study. As all other validity criteria were achieved, it is the opinion of the study director that the results obtained here may still be considered an accurate representation of the aquatic toxicity test substance to pseudokirchneriella subcapitata.

 

Kindly refer the attached background material section of the IUCLID for graphs.

Analysis

The exposure concentrations used in this study were below the minumum limits of detection and quantification achievable by the analytical method. Therefore, upon discussion with and at the request of the sponsor, the stock solution of Test substance in deionised water used to dose the test vessels was analysed at day 0 to qualify achieved concentrations. The results indicate that the measured concentration of the stock solution remained within 80-120% of the nominal concentration (actual value 102.528%). Therefore, effect concentrations are reported as the nominal concentrations of test substance as received.

Validity criteria fulfilled:

no

Remarks:

Day-by-day coefficient of variation exceeded OECD 201 guideline limits, all other validity criteria were achieved.

Conclusions:

Based on study results, the 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 0.72, 0.17, 0.27, 0.12 and 0.125 loading rate WAF mg/L respectively (i.e., equivalent to 0.367, 0.09, 0.14, 0.061 and 0.064 mg a.i./L respectively).

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, ‘Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), 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 0.1 and 1 mg/L (by growth rate as well yield). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 0.0625, 0.125, 0.25 0.5, and 1 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 80-120% of the nominal concentration. Therefore, effect concentrations were reported as the nominal as received and were expressed as loading rates, following preparation of water accommodated fractions (WAF’s). 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, 0.0625, 0.125 and 0.25 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 0.5 mg/L conical flasks appeared slightly green after 48 and 72 h and 1 mg/L conical flasks appeared clear and colourless throughout the test. The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be 0.72 (95% confidence interval: 0.53 -1.16), 0.17 (95% confidence interval: 0.04 -0.28), 0.27 (95% confidence interval: 0.22-0.32), 0.12 (95% confidence interval: 0.07-0.16) loading rate WAF mg/L (nominal), respectively. The NOEC value was determined to be 0.125 mg/L (nominal), for both growth rate and cell particle densities. The measured control cell density increase was recorded as 67.7, corresponding to a specific growth rate of 1.404 per day (which is >0.92 per day). The variation coefficient for the 72 h period was 2.42% (which is below the 7% limit) and for each time period 39.36% (which exceeded the 35% limit). As all other validity criteria were achieved, it was the opinion of the study director that the results obtained in the study may still be considered an accurate representation of the aquatic toxicity of the test substance to Pseudokirchneriella subcapitata. Under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 0.72, 0.17, 0.27, 0.12 and 0.125 loading rate WAF mg/L respectively (i.e., equivalent to 0.367, 0.09, 0.14, 0.061 and 0.064 mg a.i./L respectively) (Chemex, 2018).

Description of key information

Based on the study results, the 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 0.72, 0.17, 0.27, 0.12 and 0.125 loading rate WAF mg/L respectively (i.e., equivalent to 0.367, 0.09, 0.14, 0.061 and 0.064 mg a.i./L respectively).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.367 mg/L

EC10 or NOEC for freshwater algae:

0.09 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, ‘Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), 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 0.1 and 1 mg/L (by growth rate as well yield). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 0.0625, 0.125, 0.25 0.5, and 1 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 80-120% of the nominal concentration. Therefore, effect concentrations were reported as the nominal as received and were expressed as loading rates, following preparation of water accommodated fractions (WAF’s). 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, 0.0625, 0.125 and 0.25 mg/L conical flasks appeared clear and colourless after 24 h, slightly green after 48 h and green after 72 h. 0.5 mg/L conical flasks appeared slightly green after 48 and 72 h and 1 mg/L conical flasks appeared clear and colourless throughout the test. The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be 0.72 (95% confidence interval: 0.53 -1.16), 0.17 (95% confidence interval: 0.04 -0.28), 0.27 (95% confidence interval: 0.22-0.32), 0.12 (95% confidence interval: 0.07-0.16) loading rate WAF mg/L (nominal), respectively. The NOEC value was determined to be 0.125 mg/L (nominal), for both growth rate and cell particle densities. The measured control cell density increase was recorded as 67.7, corresponding to a specific growth rate of 1.404 per day (which is >0.92 per day). The variation coefficient for the 72 h period was 2.42% (which is below the 7% limit) and for each time period 39.36% (which exceeded the 35% limit). As all other validity criteria were achieved, it was the opinion of the study director that the results obtained in the study may still be considered an accurate representation of the aquatic toxicity of the test substance to Pseudokirchneriella subcapitata. Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 0.72, 0.17, 0.27, 0.12 and 0.125 loading rate WAF mg/L respectively (i.e., equivalent to 0.367, 0.09, 0.14, 0.061 and 0.064 mg a.i./L respectively) (Chemex, 2018).