Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

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:

26 Nov 2017 - 17 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test item concentrations and control
- Sampling method: Samples were taken from the control and all surviving test groups at 0 and 72 h from fresh media and at 24 and 96 h from old media for quantitative analysis. Duplicate samples and samples at 24 h (fresh media), 48 h (old and fresh media) and 72 h (old media) were taken
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2200 and 1100 mg of test item were dissolved in 22 and 11 L test water, respectively and stirred at 1500 rpm for 24 h. After stirring, the media was pooled to give the 100 mg/L test concentration from which a series of dilutions was made to give the remaining test concentrations. Each test preparation was mixed with a flat bladed mixer for approximately 1 min to ensure homogeneity.
- Controls: Yes

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Length at study initiation (length definition, mean, range and SD): 5.9 cm (standard deviation = 0.43)
- Weight at study end (mean and range, SD): 1.7 g (standard deviation = 0.54)
- Method of breeding: glass fiber tank with a "single pass" water renewal system, at 14 °C and dissolved oxygen content of ≥9.7 mg O2/L; 16 h light and 8 h darkness cycle with 20 min dawn and dusk transition periods.

ACCLIMATION
- Acclimation period: 26 Feb 2018 to 05 Mar 2018
- Feeding frequency during acclimation: commercial trout pellets which was discontinued approx 26 h prior to the start of the definitive test
- Health during acclimation (any mortality observed): no mortality in the 7 days prior to the start of the test

FEEDING DURING TEST : The fish were not individually identified and received no food during exposure.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

140 mg/L as CaCO3

Test temperature:

13 °C - 14 °C

pH:

7.4 - 8.2 (Control)
7.4 - 8.3 (Test item concentrations)

Dissolved oxygen:

9.3 - 11.8 mg O2/L (Control)
8.7 - 12.0 mg O2/L (Test item concentrations)

Conductivity:

345 μS/cm at 20 °C

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered to reduce evaporation)
- Material, size, headspace, fill volume: 25 to 30 L glass exposure vessels containing 20 L test volume
- Aeration: aerated via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 7 (at 32 mg/L only 6)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Based on the mean body weight value this gave a loading rate of 0.60 g body weight/liter.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was used as test water; dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Metals: Copper 0.02 mg Cu/L, Lead 0.51 µg Pb/L, Nickel 3.1 µg Ni/L, Iron 19.9 µg Fe/L
- Pesticides: 0 µg/L
- Chlorine: 0.32 mg/L
- Culture medium different from test medium: No
- pH: 7.66
- Intervals of water quality measurement: water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h darkness cycle with 20 min dawn and dusk transition periods
- Others: 13 °C to 14 °C in a temperature controlled room

EFFECT PARAMETERS MEASURED: The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 h after the start of exposure and then daily throughout the test until termination after 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Mortalities were observed at 100 mg/L.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other biological observations: See tables 1 and 2 in section "Any other information on results incl tables".
- Mortality of control: No

Reported statistics and error estimates:

The LC50 values were calculated by the Trimmed Spearman-Karber procedure. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Loading Rate (NOEC) was calculated using the Fisher’s Exact Binomial Test with Bonferroni correction and the Step-down Cochran Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Sublethal observations / clinical signs:

Biological Results:

Table 1: Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7*)							Mortality (%)
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
32	0	5	6	6	6	6	6	100
100	7	7	7	7	7	7	7	100

 

Table 2: Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration (mg/L)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7	7/7
1.0	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7	7/7
3.2	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7	7/7
10	No abnormalities detected	7/7	7/7	7/7
	Swimming at bottom				7/7	7/7	7/7	7/7
32	Swimming at bottom	6/6	1/6	AD
	Moribund		2/6
100	No abnormalities detected	A/D

*6 in 32 mg/L test group

AD  = All fish dead

Table 3: Validity criteria for OECD 203

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	No mortality was observed	Yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	≥60%	Yes

Table 4: Analytical results:

Time Point	Nominal Concentration of Test Item in Test Sample cnom	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample   c	Percentage of Nominal Concentration
[Hours]	[mg/L]		[mg/L]	[%]
0	Control	4	<LOQ	-
(Fresh)	1.0	10	0.856	86
	3.2	40	2.82	88
	10	100	8.70	87
	32	400	29.5	92
	100	800	80.9	81
24	Control	4	<LOQ	-
(Old)	1.0	10	0.871	87
	3.2	40	2.73	85
	10	100	8.67	87
	32	400	30.7	96
	100	800	82.2	82
72	Control	4	<LOQ	-
(Fresh)	1.0	10	0.847	85
	3.2	40	3.14	98
	10	100	9.36	94
96	Control	4	<LOQ	-
(Old)	1.0	10	0.925	93
	3.2	40	3.02	94
	10	100	9.63	96

LOQ              =         Limit of Quantification

-                     =         not applicable

Validity criteria fulfilled:

yes

Remarks:

see Table 3Validity criteria for OECD 203 in section "Any other information on results incl tables"

Description of key information

LC50 (96 h)= 18 mg/L (Oncorhynchus mykiss, OECD 203, nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

18 mg/L

Additional information

One experimental study is available investigating the acute toxicity of the substance to fish. The study was conducted according to OECD 203 (GLP) using rainbow trout (Oncorhynchus mykiss) as test species (Envigo, 2018). The test was performed under semi-static conditions. Five concentrations of "WAFs" (Water accommodated fractions) were performed with the nominal loading rates of 1.0, 3.2, 10.0, 32 and 100 mg/L. Analysis of the freshly prepared test preparations at 0 and 72 hours, and of the old or expired test preparation at 24 and 96 hours showed measured test concentrations between 80 and 120% of nominal. Thus it is justified that the effect concentrations are based on the nominal concentrations. Sublethal effects (swimming at bottom, moribund fish) were observed at the test item concentrations of 10 and 32 mg/L. The LC 50 for mortality after 96 h was determined to be at 18 mg/L and the NOEC at 10 mg/L.