Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2015 - August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

did not affected the quality or integrity of the study

Qualifier:

according to guideline

Guideline:

other: Guideline on bioanalytical method validation, EMEA/CHMP/EWP/192217/2009 (21 July 2011)

Deviations:

yes

Remarks:

did not affected the quality or integrity of the study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- batch No.of test material: 3706OB
- Expiration date of the lot/batch: March 2019
- Purity: >99%
- Appearance: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature at 15-25°C.

Analytical monitoring:

yes

Details on sampling:

Samples were taken for analysis from the freshly prepared solutions at 0 and 48 hours, and the expired solutions at 48 and 96 hours at all the test concentrations.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: WAF
According to the poor solubility in water of the test item, a WAF method was used in test solution preparation. 0.0500-0.0506, 0.0717-0.0718, 0.1003, 0.1399-0.1405 and 0.2000-0.2007 g of the test item were weighted and moved into five glass bottles (with a tap at the bottom side) respectively. 10 L of test water were filled into the bottles, and then be stirred on a magnetic stirrer(200 rpm) for 23 hours. After a standing time of 1 hour, the test solution was filtrated by tube filled with 5 cm glass wool. the filtered solutions were collected through the tubes (filled with 5 cm glass wool) which were connected to the taps. About 100 ml initial filtered solutions were discarded before 5 L filtered solutions of each were collected to use as test solutions.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Strain: 20140929
- Source: Shenyang Shenglongfu Laboratory Equipment Ltd
- Age at study initiation (mean and range, SD):
- Length at study initiation: within 2.4 - 2.8 cm
- Weight at study initiation : .1653 - 0.2431 g


ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions : same as test
- Type and amount of food during acclimation: fed with Artemia Salina nauplii daily until 24 hours before the test started.


FEEDING DURING TEST
no feeding during the test

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

ranged from 21.2 to 22.7°C during the test

pH:

ranged from 7.04 to 8.11 during the test

Dissolved oxygen:

ranged from 60.9 to 95.6% of the air saturation value (ASV) during the test

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Range finding test: between 5 and 30 mg/L (nominal concentration).
Definitve test: 5.0, 7.1, 10, 14 and 20 mg/L (nominal concentration).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED :
- Toxic signs and mortality were recorded at 2, 24, 48, 72 and 96 hours after exposure.
- During 96 hours test period, the dissolved oxygen, pH and temperature in test media were determined at 0, 24, 48(fresh and expired), 72 and 96 hour exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: less than 2
- Range finding study: yes, between 5 and 30 mg/L (nominal concentration).
- Test concentrations: 5.0, 7.1, 10, 14 and 20 mg/L (nominal concentration) in the definitve test

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

1) Toxicity results (see table 1, in "Any other information on results including tables) :
- Control: No death and visible toxic sign was observed
- 5 mg/L: 2 deaths
- 7.1 and 10 mg/L solutions: visible toxic signs of lethargy and erratic swimming, 6 and 7 deaths respectively
- 14 and 20 mg/L solutions: visible toxic signs of lethargy and erratic swimming and all death in 48 hours

2) Analytical results (see table 2, in "Any other information on results including tables) :
For the nominal test item concentrations of 5.0, 7.1, 10, 14 and 20 mg/L, the mean measured concentrations were 1.95, 3.05, 3.01, 4.39 and 5.33 mg/L in the fresh solutions, , the mean measured concentrations of 5.0, 7.1 and 10 mg/L nominal concentrations were 1.80, 2.75 and 2.76 mg/L in the expired solutions. The overall mean measured concentrations were 1.88, 2.91, 2.88, 4.39 and 5.33 mg/L, respectively.
The measured concentrations of expired solution were 90.2% - 92.4% of the fresh concentrations, which confirmed the stability of exposure concentrations of test media

Sublethal observations / clinical signs:

Table 1. Cumulative Mortality and Abnormalities

Nominal Concentration (mg/L)	Measured Concentration  (mg/L)	Initial Population	Time (hr)	Cumulative Mortality	Abnormalities
Control	2	-	0	10	No poisoning signs
	24		0		No poisoning signs
	48		0		No poisoning signs
	72		0		No poisoning signs
	96		0		No poisoning signs
5.0	1.88	10	2	0	No poisoning signs
			24	0	No poisoning signs
			48	0	No poisoning signs
			72	1	1Erratic swimming, 8 no poisoning signs
			96	2	8 no poisoning signs
7.1	2.91	10	2	0	No poisoning signs
			24	0	No poisoning signs
			48	1	1 lethargy,  8no poisoning signs
			72	4	3 erratic swimming, 3no poisoning signs
			96	6	1 erratic swimming, 3no poisoning signs
10	2.88	10	2	0	No poisoning signs
			24	0	1lethargy, 9 no poisoning signs
			48	1	1 lethargy, 8no poisoning signs
			72	3	4 erratic swimming, 3no poisoning signs
			96	7	3no poisoning signs
14	4.39	10	2	0	No poisoning signs
			24	2	1 lethargy, 8erratic swimming& lethargy
			48	10	-
20	5.33	10	2	0	No poisoning signs
			24	8	2 lethargy &erratic swimming
			48	10	-

Table 2: Concentrations of Test Item in the Exposure Solution

Nominal Concentration (mg/L)	Replicate	Mean Measured Concentration (mg/L)				Mean Fresh (mg/L)	Mean Expired (mg/L)	Overall Mean (mg/L)	Expired/ Fresh^1 (%)
		02/09/2015	02/11/2015		02/13/2015
		0h-Fresh	48h-Expired	48h-Fresh	96h-Expired
Control	1	N/A	N/A	N/A	N/A	N/A	N/A	N/A	-
5.0	1	1.85	1.63	2.04	2.00	1.95	1.80	1.88	92.4%
	2	1.83	1.68	2.04	1.95
	3	1.86	1.76	2.08	1.79
	Mean	1.85	1.69	2.05	1.91
7.1	1	2.98	N/A	3.20	2.90	3.05	2.75	2.91	90.2%
	2	2.92	2.52	3.12	2.88
	3	2.92	2.70	3.14	2.74
	Mean	2.94	2.61	3.15	2.84
10	1	2.86	2.40	3.14	3.08	3.01	2.76	2.88	91.6%
	2	2.90	2.52	3.14	3.06
	3	2.86	2.44	3.16	3.04
	Mean	2.87	2.45	3.15	3.06
14	1	4.38	-	-	-	4.39	-	4.39	-
	2	4.44	-	-	-
	3	4.36	-	-	-
	Mean	4.39	-	-	-
20	1	5.38	-	-	-	5.33	-	5.33	-
	2	5.28	-	-	-
	3	5.34	-	-	-
	Mean	5.33	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

According to the mortality data of test, the 96-hour LC50 of XA31 to the zebra fish was 2.52 mg/L with the 95% confidence limit 2.15-2.95 mg/L, calculated as measured concentrations of test item.

Executive summary:

The acute toxicity of the Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) to zebra fish (danio rerio) was investigated using semi-static conditions (solutions replaced every 48 hours) over a 96-hour exposure period. This study was performed according to the OECD guideline 203 (Fish, Acute toxicity test) and according to the EU Method C.1 (Acute toxicity for fish) and was compliant with the GLP.

 The definitive test was conducted using the test item concentrations of 5.0, 7.1, 10, 14 and 20 mg/L (nominal concentrations). A concurrent blank (test media) control was performed in parallel. Ten fish were used for each of the test groups and the control group.

During the 96-hour exposure period, no death and visible toxic sign was observed in the control vessels. 2 deaths were observed in the 5.0 mg/L exposed test group. Fish in the 7.1 and 10 mg/L solution were observed visible toxic signs of lethargy and erratic swimming and finally 6 and 7 of them dead respectively. In test vessels with the concentrations of 14 and 20 mg/L, the fish showed visible toxic signs of lethargy and erratic swimming and death.

During the test period, the results showed that for the nominal test item concentrations of 5.0, 7.1, 10, 14 and 20 mg/L, the mean measured concentrations were 1.95, 3.05, 3.01, 4.39 and 5.33 mg/L in the fresh solutions and the overall mean measured concentrations were1.88, 2.91, 2.88, 4.39 and 5.33 mg/L,respectively. The measured concentrations of expired solution were 90.2% - 92.4% of the fresh concentrations, which confirmed the stability of exposure concentrations of test media.

According to the mortality data of test, the 96-hour LC50of Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) to the zebra fish was 2.52 mg/L with the 95% confidence limit 2.15-2.95 mg/L, calculated as measured concentrations of test item.

The three validity criteria of the OECD guideline 203 were fulfilled, therefore this study is considered as reliable without restrictions.

Description of key information

One reliable study is available for the Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) for this endpoint.

The acute toxicity (EC50) of the Reaction mass of AminoPhosphonium salt and Bisphenol AF towards fish (Danio rerio) was determined in a 96‑hour semi-static test according to the for the Testing of Chemicals No. 203 “Fish, Acute Toxicity Test” (Adopted: 17th July 1992)" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008. The study was compliant with the GLP.

Due to the low aqueous solubility and complex nature of the test item, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), thus all the test solutions were prepared as a Water Accommodated Fraction (WAF). 

Based on a preliminary range-finding test, in the definitive test fish were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates conducted of 5.0, 7.1, 10, 14 and 20 mg/L. A concurrent blank (test media) control was performed in parallel. Ten fish were used for each of the test groups and the control group.

During the test period, the results showed that for the nominal test item concentrations of 5.0, 7.1, 10, 14 and 20 mg/L, the mean measured concentrations were 1.95, 3.05, 3.01, 4.39 and 5.33 mg/L in the fresh solutions and the overall mean measured concentrations were1.88, 2.91, 2.88, 4.39 and 5.33 mg/L,respectively. The measured concentrations of expired solution were 90.2% - 92.4% of the fresh concentrations, which confirmed the stability of exposure concentrations of test media.

During the 96-hour exposure period, no death and visible toxic sign was observed in the control vessels. 2 deaths were observed in the 5.0 mg/L exposed test group. Fish in the 7.1 and 10 mg/L solution were observed visible toxic signs of lethargy and erratic swimming and finally 6 and 7 of them dead respectively. In test vessels with the concentrations of 14 and 20 mg/L, the fish showed visible toxic signs of lethargy and erratic swimming and death.

According to the mortality data of test, the 96-hour LC50of Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) to the zebra fish was 2.52 mg/L with the 95% confidence limit 2.15-2.95 mg/L, calculated as measured concentrations of test item.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.52 mg/L