[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 7, 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 Chemical (second version), "No.202 'Daphnia sp. Acute Immobilization Test'"

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic acids, C16-alkane hydroxy and C16-alkene, potassium salts

Cas Number:

2210238-42-7

IUPAC Name:

Sulfonic acids, C16-alkane hydroxy and C16-alkene, potassium salts

Test material form:

cream / paste

Remarks:

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

Details on test material:

pH: 9.0-10.5 (diluted 10% act. solution)
Stability: Stable in container after opening. Stable in water and in light. Stability in soil is
not applicable

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:

When the test started (0h) and the test finished (48h), took 1.0 mL every test solution and diluted it with deionized water, then determined the actual concentration of these test solutions by the analytical method for concentration.

Test solutions

Vehicle:

no

Details on test solutions:

The water used for the test was tap-water after aeration and eliminating chlorine. The physicochemical parameters of the water were: pH was 6.9; total hardness was 140 - 250 mg/L CaCO3, the DO was greater than 80% ASV.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms
The test organisms were Daphnia magna Straus which were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences in April 2018 and it was cultured in the laboratory.

Daphnia Feeding
The tap water which was aerating and eliminating chlorine was put into the 2000 mL beaker. Dozens of Daphnia were added and fed it with cultivated algae of mine lab. The culture temperature was 20 士 2°C; light cycle was 16-hour light and 8-hour dark cycle. Illumination intensity was 1000~1500 Lux. The growth of Daphnia was observed and the young Daphnia were removed out in time every day. The test Daphnia was Daphnia magna which of the age were less than 24h, and the generation was not the fast generation.

Collection of Daphnia
The day before the test started, the young Daphnia were separated from mother Daphnia in incubator. The young Daphnia which of the generation was not the first generation and the age was less than 24h could be obtained on the second day. This Batch of Daphnia was D201903031229.

Study design

Test type:

static

Water media type:

other: Tap water

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

140~250 mg/L CaCO3

Test temperature:

20±2℃

pH:

6~9

Dissolved oxygen:

>80%

Nominal and measured concentrations:

Nominal concentrations: 11, 15, 20, 27, 37, 50 mg active ingredient/L

Measured concentrations at 0 h: 11.45, 16.25, 19,21, 28.17, 40.17, 48.94 mg active ingredient/L
Measured concentrations at 48 h: 10.83, 14.58, 16.24, 25.74, 35.12, 45.71 mg active ingredient/L

Details on test conditions:

Preliminary Test
The nominal concentrations such as 1, 5, 10, 50, 100 mg a.i./L of C16K test solution and blank control were prepared in preliminary test. 50 mL test solution was added to each beaker. Every group added 5 young Daphnia which of the age were less than 24h and the generation was more than the first generation. The static test method was selected to perform preliminary test, the test period was 48h.
The inhibition of Daphnia was observed and recorded at 0h, 24h and 48h. The dissolved oxygen concentration, pH was measured at 0h and 48h. Took a cup 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 18.9 and 20.3 ℃.

Definitive test
According to test results of the preliminary and method stability test, the nominal concentrations such as 11, 15, 20, 27, 37, 50 mg a.i./L test solution of C16K and blank control were prepared to perform definitive test. The static test method was selected to perform definitive test, and the test period was 48h.
Precisely weighed 0.0941 g test substance C16K and added it into a 100 mL beaker, dissolved it with test water (pH=5.9 土 0.05) and transferred it into a 500 mL volumetric flask and made constant volume to scale with test water, it was ultrasonic dissolved for 10 min and constantly, finally the nominal concentration of 100 mg a.i./L stock solution was obtained.
27.5, 37.5, 50.0, 67.5, 92.5, 125.0 mL stock solution which nominal concentration was 100 mg a.i./L of C16K were added into each 250 mL volumetric flask and made constant volume to scale with test water, then the test solutions which nominal concentrations were 11, 15, 20, 27, 37, 50 mg a.i./L were prepared. Every beaker added 50 mL test solution, four parallel control groups and a blank control group while be set in definitive test.
Every beaker added 5 Daphnia which the age was less than 24h and the generation was more than the first generation. The inhibition of Daphnia was observed and recorded at 0h, 24h and 48h. The dissolved oxygen concentration, pH and the actual concentration of the test solution were measured at 0h and 48h. Took a cup of test solution into the incubator which as same as blank group to record the temperature every day.

Data Analysis
The value of ECx and 95% limit confidence was statisticed with EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES VERSION 1.5.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

23.86 mg/L

95% CI:

21.09 - 27.12

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19.19 mg/L

95% CI:

17.03 - 21.59

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Preliminary Test
The inhibition of Daphnia was observed and recorded at 0h, 24h and 48h. The dissolved oxygen concentration, pH was measured at 0h and 48h. Took a cup 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 18.9 and 20.3 ℃. The results of preliminary test were shown below in table 2 and table 3.

Definitive test
The temperature of the cup of test solution was between 19.3 and 20.7 ℃.
The results of definitive test were given in table 4 and table 5.

Test Validity
(1) At the end of the test, the dissolved oxygen of control and test solution were greater than 3.0 mg/L.
(2) At the end of the test, the inhibition rate for Daphnia in the control group is 0, it was not more than I0%.
(3) During the test period, the actual concentration of test solution was greater than 80% of its original measured concentration
(4) The 24h-EC50 of potassium dichromate to Daphnia magna was 1.077 mg/L, the 95% limit confidence was 0.965~1.210 mg/L, the sensitivity of Daphnia magna was compliant with the requirements of guidelines.

Results with reference substance (positive control):

Sensitivity test
The recent sensitivity test of Daphnia conducted on Feb 19th, 2019. Potassium dichromate was used as the reference substance to carry out the sensitivity test. Reference test result showed that: the Daphnia inhibition ratio was 0 in blank control, and the 24h-EC50 of potassium dichromate on Daphnia was 1.077 mg/L, the 95% limit confidence was 0.965~1.210 mg/L. 24h-EC50 was between 0.6 and 2.1 mg/L, the sensitivity of Daphnia was compliant with the requirements of guidelines.

Any other information on results incl. tables

Table 2 Results of Daphnia Inhibition Quantity in preliminary test

Nominal concentration (mg a.i./L)		0	1	5	10	50	100
Quantity of Daphnia		5	5	5	5	5	5
Inhibition Quantity	0h	0	0	0	0	0	0
	24h	0	0	0	0	5	5
	48h	0	0	0	0	5	5

Table 3 pH and Dissolved Oxygen result s in preliminary test

Nominal concentration  (mg a.i./L)	0h		48h
	pH	Dissolved Oxygen (mg/L)	pH	Dissolved Oxygen (mg/L)
0	6.00	8.05	7.44	7.88
1	6.10	7.89	7.52	7.62
5	6.08	8.04	7.49	7.49
10	6.09	7.85	7.48	7.45
50	6.22	7.64	7.44	7.06
100	6.26	7.16	7.42	6.93

Table 4 Results of Daphnia Inhibition Quantity and Toxicity Symptom in definitive test

Nominal concentration (mg a.i./L)	Quantity of Daphnia	0h		24h		48h
		Inhibition Quantity	Toxicity Symptom	Inhibition Quantity	Toxicity Symptom	Inhibition Quantity	Toxicity Symptom
0	5	0	A(5)	0	A(5)	0	A(5)
	5	0	A(5)	0	A(5)	0	A(5)
	5	0	A(5)	0	A(5)	0	A(5)
	5	0	A(5)	0	A(5)	0	A(5)
11 (Actual Value was 11.41)	5	0	A(5)	0	A(5)	0	A(5)
	5	0	A(5)	0	A(5)	1	A(4) E(1)
	5	0	A(5)	0	A(5)	0	A(5)
	5	0	A(5)	0	A(5)	0	A(5)
15 (Actual Value was 15.39)	5	0	A(5)	0	A(5)	1	A(4) F(1)
	5	0	A(5)	1	A(4) E(1)	2	A(3) E(1) F(1)
	5	0	A(5)	1	A(4) F(1)	1	A(4) F(1)
	5	0	A(5)	1	A(4) E(1)	1	A(4) F(1)
20 (Actual Value was 17.67)	5	0	A(5)	2	A(3) F(2)	3	A(1) B(1) F(3)
	5	0	A(5)	1	A(4) F(1)	2	A(3) E(1) F(1)
	5	0	A(5)	2	A(3) F(2)	3	A(2) F(3)
	5	0	A(5)	2	A(3) E(1) F(1)	3	A(2) F(3)
27 (Actual Value was 26.93)	5	0	A(5)	4	A(1) E(1) F(3)	4	B(1) F(4)
	5	0	A(5)	3	A(2) E(1) F(2)	4	A(1) F(4)
	5	0	A(5)	3	A(2) F(3)	4	A(1) E(1) F(3)
	5	0	A(5)	2	A(3) F(2)	3	A(2) F(3)
37 (Actual Value was 37.56)	5	0	A(5)	4	B(1) E(1) F(3)	5	F(5)
	5	0	A(5)	3	B(2) F(3)	5	F(5)
	5	0	A(5)	3	B(2) F(3)	5	F(5)
	5	0	A(5)	5	B(3) F(2)	4	B(1) F(4)
50 (Actual Value was 47.30)	5	0	A(5)	5	F(5)	5	F(5)
	5	0	A(5)	5	F(5)	5	F(5)
	5	0	A(5)	5	F(5)	5	F(5)
	5	0	A(5)	5	F(5)	5	F(5)
EC50 (mg a.i./L)	-	-		23.86		19.19
95% limit confidence (mg a.i./L)	-	-		21.09～27.12		17.03～21.59
A-Normal, B-Swimming slowly, C-Anomalous fast swimming, D-Change of body coloration, E-Those animals that are not to swim within 15 seconds after gentle agitating of the test vessel, F-died.

Table 5 pH and Dissolved Oxygen result s in definitive test

Nominal concentration (mg a.i./L)	Oh		48h
	pH	Dissolved Oxygen (mg/L)	pH	Dissolved Oxygen (mg/L)
0	6.03	7.87	7.09	7.53
11	6.07	7.47	7.19	7.59
15	6.13	7.29	7.23	7.66
20	6.12	7.23	7.23	7.68
27	6.06	7.18	7.23	7.70
37	6.09	7.21	7.24	7.67
50	6.15	7.17	7.28	7.62

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The result of the definitive test indicated that: The 24h-EC50 of C16K on Daphnia magna in test solution was 23.86 mg a.i./L (actual concentration), 95% limit confidence was 21.09~27.12 mg a.i./L (actual concentration); The 48 h-EC50 of C16K on Daphnia magna in test solution was 19.19 mg a.i./L (actual concentration), 95% limit confidence was 17.03~21.59 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" No. 202 'Daphnia sp. Acute Immobilization Test(Second edition)' and "OECD Guidelines for Testing of Chemicals No.202 'Daphnia sp. Acute Immobilization Test"'. According to the study plan and relevant SOP, Daphnia sp. Acute Immobilization Test of C16K was performed.

 According to test results of the preliminary and method stability test, the nominal concentrations such as 11, 15, 20, 27, 37, 50 mg a.i./L test solution of C16K and blank control were prepared to perform definitive test. The static test method was selected to perform definitive test, and the test period was 48h. Four parallel control groups and a blank control group while be set in definitive test, 50 mL test solution was added to each beaker, and every beaker added 5 Daphnia which the age was less than 24h and the generation was more than the first generation. The inhibition of Daphnia was observed and recorded at 0h, 24h and 48h. The dissolved oxygen concentration, pH and the actual concentration of the test solution were measured at 0h and 48h. Took a cup 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 19.3 and 20.7 ℃.

 The concentration determination result showed that, in the test period (48h), the test solutions which of the nominal concentration of the test substance C16K were 11, 15, 20, 27, 37, 50 mg a.i./L, the actual concentrations were 11.14, 15.39, 17.67, 26.93, 37.56, 47.30 mg a.i./L.

 The result of the definitive test indicated that: The 24h-EC50 of C16K on Daphnia magna in test solution was 23.86 mg a.i./L (actual concentration), 95% limit confidence was 21.09~27.12 mg a.i./L (actual concentration); The 48h-EC50 of C16K on Daphnia magna in test solution was 19.19 mg a.i./L (actual concentration), 95% limit confidence was 17.03~21.59 mg a.i./L (actual concentration) .