[No public or meaningful name is available]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 28, 2019 to April 10, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004, "The Guidelines for the Testing of Chemicals" of " No. 201 'Alga Growth Inhibition Test'" (Second edition)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic Acids, C18-alkane hydroxyl and C18-alkene, potassium salts

Cas Number:

2210238-56-3

IUPAC Name:

Sulfonic Acids, C18-alkane hydroxyl and C18-alkene, potassium salts

Test material form:

cream / paste

Remarks:

Pale yellow paste at 50℃, This sample was obtained from the original liquid form by removing water.

Specific details on test material used for the study:

Storage Condition: Store container tightly closed in well-ventilated place. Store in corrosive resistant/container with a resistant in liner. Store locked up. Don ' t store with acids.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Took 1.0 mL of 25, 50 and 100 mg a.i./L test solution in 10 mL volumetric flask at 0, 24, 48 and 72h, dilute with deionized water to scale, blending, and took 1.0 mL those above solution into a 100 mL volumetric flask, dilute with deionized water to scale, took test solution after blending, measured its concentration under instrument condition. Took 1.0 mL of 200, 400 and 800 mg a.i./L test solution in 50 mL volumetric flask at 0,24, 48 and 72h, dilute with deionized water to scale, blending, and took 1.0 mL those above solution into 100 mL volumetric flask, dilute with deionized water to scale, took test solution after blending, measured its concentration under instrument condition.

Test solutions

Vehicle:

no

Details on test solutions:

Culture medium was prepared with deionized water ; the final concentration of each nutritive salt in culture medium was shown as the following:
Alimentation salt Concentration
NH4Cl 15 mg a.i./L
MgCl2·6H2O 12 mg a.i./L
CaCl2-2H2O 18 mg a.i./L
MgS04·7H2O 15 mg a.i./L
KH2PO4 1.6 mg a.i./L
FeCl3·6H2O 80 µg/L
Na2EDTA·2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2-4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2·6H2O 1.5 µg/L
CuCl2·2H2O 0.01 µg/L
NaMoO4·2H2O 7 µg/L
NaHCO3 50 mg a.i./L

Test organisms

Test organisms (species):

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

Details on test organisms:

The test organisms
Selenastrum capricornutum were obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology in April 2018 and were cultivated in the laboratory.

Preliminary culture
Took a certain amount of Selenastrum capricornutum from stock culture, inoculated them in fresh and aseptic culture medium, and cultivated them under the same condition as test requirements. Selenastrum capricornutum would be inoculated in the fresh and aseptic culture medium once every 2~4 days, duplicated the same test for 2~3 times. Inspected that Selenastrum capricornutum were growing well and under logarithm growth period, thus the algae test solution was prepared for using.
The serial numbers of this test organisms as follow: S20190301090

Study design

Test type:

static

Water media type:

other: deionized water

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21~24℃

pH:

7.41~8.36

Nominal and measured concentrations:

Nominal concentrations: 25, 50, 100, 200, 400, 800 mg a.i./L
Measured concentrations 0h: 28.83, 53.71, 107.65, 209.45, 426.9, 869.9 mg a.i./L
Measured concentrations 24h: 26.06, 49.96, 103.43, 207.8, 398.4, 881.4 mg a.i./L
Measured concentrations 48h: 23.86, 48.65, 101.81, 187.0, 354.8, 752.65 mg a.i./L
Measured concentrations 72h: 23.77, 47.38, 93.00, 189.05, 355.45, 762.85 mg a.i./L
Geometric mean concentrations: 25.56, 49.87, 101.33, 198.06, 382.69, 814.55 mg a.i./L

Details on test conditions:

The nominal concentrations such as 1, 10, 100, 200 and 400 mg a.i./L of C18K test solution and blank control were prepared in preliminary test. The static test method was selected to perform preliminary test, the test period was 72h. Observed and recorded the growth of Selenastrum capricornutum in test solution of 24h, 48h and 72h. Illumination intensity and pH values of the test solutions were determined at 0h, 72h. Took a bottle of test solution into the incubator which as same as blank group to record the temperature every day. The temperature of the cup of test solution was between 22.6 and 23.5℃.


-Preparation of testing solution
Weighed 2.5575 g test substance and put it in a 100 mL beaker, added test culture medium to dissolved and transferred it to a 500 mL volumetric flask, and made constant volume to the scale with test medium, took ultrasound for 10 min, obtain the stock solution with concentration of 2000 mg a.i./L, diluted the stock solution with test medium solution to a concentration twice that of the needed test solution. The preparation of test solutions was showed in table 1.

-Operating of test
According to the preliminary test results, the nominal concentrations such as 25, 50, 100, 200, 400 and 800 mg a.i./L test solution of C18K was prepared to perform definitive test, every concentration set three parallel and a blank control. The static test method was selected to perform definitive test, the test period was 72h. Observed and recorded the growth of Selenastrum capricornutum in test solution of 24h, 48h and 72h. Illumination intensity, pH values and the actual concentration of the test solutions were determined at 0h, 72h. Took a bottle of test solution into the incubator which as same as blank group to record the temperature every day. The temperature of the cup of test solution was between 22.4 and 23.3℃.


(1) Since the initial of the test, the concentration of the alga cells increased up to 3.83 x 106 cells/mL from 2 x I04 cells/mL, increased by 192 times in 72h.
(2) The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures was less than 35%.
(3) In replicate control cultures , the coefficient of variation of average specific growth rates was less than or equal to 7% during the whole test period.



-Percent inhibition of growth rate
Calculate the percent inhibition of growth rate for each treatment replicate from equation [1]:
Ir = ( μc - μt ) / μc × 100・・・・・・[1]
Where:
Ir: Percent inhibition in average specific growth rate;
μc: Mean value for average specific growth rate (µ) in the control group;
µt: Average specific growth rate for the treatment replicate.

The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments [2]:
µi-j = ( InXj - InXi) / ( tj - ti ) ・・・・・・[2]
Where:
μi-j: The average specific growth rate from time i to j,
Xi: The biomass at time i,
Xi: The biomass at time j

-Percent inhibition of yield
The percent inhibition in yield (Iy) may be calculated for each treatment replicate as follows:
Iy = ( Yc - Yt ) / Yc × 100・・・・・・[3]
Where:
ly: percent inhibition of yield,%,
Yc: mean value for yield in the control group,
Yt: value for yield for the treatment replicate .

-Estimates of ECx and confidence limits
The ECx and 95% confidence limits can be calculated by SPSS 16.0.

-LOEC and NOEC
For estimation of the LOEC and hence the NOEC (p < 0.05), for effects of the test substance on growth rate, it is necessary to compare treatment means using analysis of variance (ANOVA) techniques. Dunnett'c was used (One-Way ANOVA, SPSS 16.0).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

49.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

589.6 mg/L

95% CI:

427.44 - 928.18

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

604.42 mg/L

95% CI:

436.24 - 959.75

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

618.34 mg/L

95% CI:

446.02 - 983.37

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

53.56 mg/L

95% CI:

18.95 - 93.26

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Effect concentrations 7

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

93.34 mg/L

95% CI:

42.27 - 165.06

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Effect concentrations 8

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

335.61 mg/L

95% CI:

186.52 - 1 021.48

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Details on results:

The test results of definitive test were given in table 4, table 5 and table 6.
Growth curves of biomass versus time of treatment group results in definitive test and Concentration-Response Curves of definitive test were given in Figure 1 and Figure 2 in attached document.

Any other information on results incl. tables

Table 2 Results of preliminary test

Nominal concentration  (mg a.i./L)	Density of algae cells (cells/mL)				Inhibition ratio (%)
	Oh	24h	48h	72h	72h-Ir	72h-Iy
0	20000	103000	541000	2890000	-	-
1	20000	107000	583000	3130000	-1.6	-8.3
10	20000	101000	515000	2620000	2.0	9.3
100	20000	66600	226000	783000	26.3	72.9
200	20000	58700	168000	474000	36.4	83.6
400	20000	58700	158000	419000	38.8	85.5

Table 3 Physical and chemical data results of preliminary test

Nominal Concentration (mg a.i./L)	pH values		Morphological observation of algae cells
	0h	72h	24h	48h	72h
0	8.05	7.92	A	A	A
1	8.03	7.45	A	A	A
10	8.03	7.42	A	A	A
100	8.07	7.72	C	C	C
200	8.14	7.82	C	C	C
400	8.47	7.94	C	C	C
A - Normal morphology         B - Cells became small               C - Paramorphia D - Color changes                 E - Adhesion aggregation

Table 4 Control group Results of definitive test

Density of algae cells (cells/mL)	0h (N)	24h (N)	48h (N)	72h (N)
	20000	112000	639000	3520000
	20000	116000	668000	3850000
	20000	125000	724000	4120000
The specific growth rate (day-1)	-	1.723	1.741	1.706
		1.758	1.751	1.752
		1.833	1.756	1.739
The average specific growth rate of coefficient of variation (%)		3.17 (0~1)d	0.44 (1~2)d	1.37 (2~3)d
The mean of coefficient of variation (%)		1.66
The average specific growth rate of each parallel (%)		1.723 (Repeat 1)	1.754 (Repeat 2)	1.776 (Repeat 3)
Coefficient of variation of the average specific growth rate (%)		1.52

Table 5 Treatment group Results of definitive test

Nominal concentration (mg a.i./L)	Density of algae cells (cells/mL)				Inhibition ratio (%)
	Oh(N)	24h (N)	48h(N)	72h(N)	24h-Ir	48h-Ir	72h-Ir	24h-Iy	48h-Iy	72h-Iy
0	20000	112000	639000	3520000	-	-	-	-	-	-
	20000	116000	668000	3850000
	20000	125000	724000	4120000
25	20000	113000	591000	3190000	3.2	3.7	3.7	5.4	12.3	17.7
	20000	103000	565000	2960000
	20000	118000	626000	3310000
50	20000	91000	407000	1780000	15.7	16.1	16.1	24.3	43.4	57.1
	20000	86100	368000	1540000
	20000	90100	375000	1610000
100	20000	74400	271000	960000	25.9	26.3	26.6	36.8	60.2	75.3
	20000	78300	271000	960000
	20000	70500	237000	895000
200	20000	62600	195000	568000	35.6	35.9	36.7	46.8	71.6	85.5
	20000	66600	212000	605000
	20000	57800	169000	498000
400	20000	62600	197000	513000	39.1	39.2	39.4	49.9	74.7	87.4
	20000	58700	168000	487000
	20000	55400	148000	449000
800	20000	47500	113000	268000	51.6	52.4	52.7	59.9	84.2	93.7
	20000	47000	98900	208000
	20000	47000	109000	247000

Table 6 Physical and chemical data result s of definitive test

Nominal concentration (mg a.i./L)	pH values		Morphological observation of algae cells
	0h	72h	24h	48h	72h
0	7.93	7.44	A	A	A
25	7.98	7.41	A	A	A
50	8.06	7.46	C	C	C
100	8.14	7.64	C	C	C
200	8.23	7.82	C	BC	BC
400	8.33	7.89	BC	BC	BC
800	8.36	8.04	BC	BC	BC
A - Normal morphology              B - Cells became small                 C – Paramorphia D - Color changes                      E - Adhesion aggregation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The result of definitive test indicated that: Alga cells concentration of blank control increased by 192 times within 72h. The 24h-EyC50 of C18K to Selenastrum capricornutum was 335.61 mg a.i./L (actual concentration), 95% confidence limit was 186.52~1021.48 mg a.i./L; the 48h-EyC50 of C18K to Selenastrum capricornutum was 93.34 mg a.i./L (actual concentration), 95% confidence limit was 42.27~165.06 mg a.i./L; the 72h-EyC50 of C18K to Selenastrum capricornutum was 43.56 mg a.i./L (actual concentration), 95% confidence limit was 18.95~94.26 mg a.i./L;
The 24h-ErC50 of C18K to Selenastrum capricornutum was 618.34 mg a.i./L (actual concentration), 95% confidence limit was 446.02~983.37 mg a.i./L; the 48h-ErC50 of C18K to Selenastrum capricornutwn was 604.42 mg a.i./L (actual concentration), 95% confidence limit was 436.24~959.75 mg a.i./L; the 72h-ErC50 of C18K to Selenastrum capricornutum was 589.60 mg a.i./L (actual concentration), 95% confidence limit was 427.44.79~928.18 mg a.i./L.
The LOEC of C18K to Selenastrum capricornutum was less than 49.87 mg a.i./L (actual concentration), and the NOEC of C18K to Selenastrum capricornutum was greater than 25.56 mg a.i./L (actual concentration).

Executive summary:

The testing of the test item was carried out according to the test method "The Guidelines for the Testing of Chemicals" of "No.201 'Alga growth inhibition test"' (Second edition) and "OECD Guidelines for Testing of Chemicals No.201 'Alga Growth Inhibition Test"', According to the study plan and relevant SOP, Alga growth inhibition test of C18K was performed with static method, the test period was 72h.

 According to the preliminary test results, the nominal concentrations such as 25, 50, 100, 200, 400 and 800 mg a.i./L test solution of C18K was prepared to perform definitive test, every concentration set three parallel and a blank control. Observed and recorded the growth of Selenastrum capricornutum in test solution of 24h, 48h and 72h. Illumination intensity, pH values and the actual concentration of the test solutions were determined at 0h, 72h. Took a bottle of test solution into the incubator which as same as blank group to record the temperature every day. The temperature of the cup of test solution was between 22.4 and 23.3℃.

The result of definitive test indicated that: Alga cells concentration of blank control increased by 192 times within 72h. The 24h-E_yC₅₀ of C18K to Selenastrum capricornutum was 335.61 mg a.i./L (actual concentration), 95% confidence limit was 186.52~1021.48 mg a.i./L; the 48h-E_yC₅₀ of C18K to Selenastrum capricornutum was 93.34 mg a.i./L (actual concentration), 95% confidence limit was 42.27~165.06 mg a.i./L; the 72h-E_yC₅₀ of C18K to Selenastrum capricornutum was 43.56 mg a.i./L (actual concentration), 95% confidence limit was 18.95~94.26 mg a.i./L;
The 24h-E_rC₅₀ of C18K to Selenastrum capricornutum was 618.34 mg a.i./L (actual concentration), 95% confidence limit was 446.02~983.37 mg a.i./L; the 48h-E_rC₅₀ of C18K to Selenastrum capricornutwn was 604.42 mg a.i./L (actual concentration), 95% confidence limit was 436.24~959.75 mg a.i./L; the 72h-E_rC₅₀ of C18K to Selenastrum capricornutum was 589.60 mg a.i./L (actual concentration), 95% confidence limit was 427.44.79~928.18 mg a.i./L.
The LOEC of C18K to Selenastrum capricornutum was less than 49.87 mg a.i./L (actual concentration), and the NOEC of C18K to Selenastrum capricornutum was greater than 25.56 mg a.i./L (actual concentration).