Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

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 January 2018 to 16 October 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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98%; Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Yellow Liquid

Analytical monitoring:

yes

Details on sampling:

Aqueous samples (8 mL) were collected from the control and each surviving test concentration at 0 (fresh media) and 96 hours (old media) for analysis. The aqueous samples were diluted with acetonitrile, then diluted further with diluent (treated tap water:acetonitrile, 4:1, v/v) as required to bring the response within the calibration range. Duplicate samples were collected, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

yes

Details on test solutions:

Preliminary analysis in another study with this UVCB test substance indicated that the test substance was soluble in aqueous test media. Test solution preparation followed the method developed in that study. At the start of the test, the test substance was weighed onto a glass slide and placed in treated tap water. The preparation was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparation was allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 100 mg/L test concentration. The stock solutions were diluted with treated tap water to give the remaining test concentrations. The control solution consisted only of treated tap water.

At the start of the test, the 100 mg/L test concentration was observed to be a slightly cloudy white homogenous dispersion that omitted an odour. The control and remaining test concentrations were observed to be colourless solutions with no odour. The test preparations were observed to be colourless solutions throughout the duration of the test. From 24 hours onwards for the remainder of the test, the control and all test concentrations were observed to be colourless solutions with no odour.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Northern Trout (Hebden, Skipton, North Yorkshire, United Kingdom)
- Length at study initiation): 4.5 cm (mean); 3.8 to 5.2 cm
- Weight at study initiation: 0.8 g (mean); 0.42 to 1.27 g

ACCLIMATION
- Acclimation period: 20 days
- Acclimation conditions: Same as test conditions
- Type and amount of food during acclimation: Fish were fed during acclimation with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish. Uneaten food and debris was siphoned or cleaned from the tanks as required.
- Health during acclimation (any mortality observed): 0% mortality observed in the stock batch of fish in the seven days prior to test initiation.

FEEDING DURING TEST
The fish were not fed during the 24 hours before the start of the definitive test and were not fed throughout the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

82 mg/L as CaCO3

Test temperature:

15 ± 2 ºC

pH:

6.65 to 7.91

Dissolved oxygen:

9.49 to 11.28 mg/L (>60% saturation)

Salinity:

Not applicable

Conductivity:

680 µS/cm

Nominal and measured concentrations:

Nominal: 0 (control), 10, 18, 32, 56 and 100 mg/L
Measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 L constructed glass aquaria, each fitted with a clear plastic lid
- Volume of test media: 10 L
- Aeration: Yes; sufficient to maintain oxygen concentration
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 0.56 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Treated tap water; passed through activated carbon filters.
- Alkalinity: 18.4 mg/L (as CaCO3)
- Ca/mg ratio: 6
- Culture medium different from test medium: Same
- Intervals of water quality measurement: Dilution water is analyzed bi-annually; test media pH, concentration of dissolved oxygen (% air saturation value and mg/L) and temperatures were recorded daily; continuous temperatures were measured using a digital (min/max) thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hour light: 8 hours dark lighting cycle with a 30 minute transition period
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Fish in each test vessel were observed for mortality and sublethal effects at ca 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test.

TEST CONCENTRATIONS
Analysis of the freshly prepared media at 0 hours resulted in measured concentrations of 64% to 71% of nominal value. The initial 0-hour results passed analytical batch acceptance criteria, but were below 80% of nominal, so the duplicate 0-hour samples were analysed alongside the 96-hour samples. Analysis of the samples collected 96 hours resulted in measured concentrations of 63% to 94% and analysis of the duplicate 0-hour samples confirmed the initial results; however, the initial 96-hour and duplicate 0-hour sample analysis failed the analytical batch acceptance criteria.

The chromatography column was changed to address this issue, but it was not possible to re-establish the chromatography seen previously due to apparent differences in performance between the old and new columns. Results from the range-finding test, although generated using the method before it was fully validated, indicated that the test substance would be stable in test media over a 96-hour period. As the initial 0-hour results were considered valid, but were below 80% of nominal value, the results from the definitive exposure were based on the initial 0-hour measured concentrations: 7.1, 12, 22, 38 and 71 mg/L.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

(95% confidence limits, 15 – 21 mg/L)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The LC50 values and corresponding 95% confidence limits at 24, 48, 72 and 96 hours were calculated using the Spearman-Kärber method. The toxicity results were expressed in terms of initial 0-hour measured concentrations. The NOEC and LOEC values were established by observation of the data.

Sublethal observations / clinical signs:

Summary of fish mortality and sublethal effects during the definitive test

Initial 0-hour measured concentration (mg/L)	Cumulative number of mortalities recorded*
	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0
7.1	0	0	0	0
12	0	0**	0**	0**
22	6***	6	6	6
38	7	7	7	7
71	7****	7	7	7

* Seven fish initially added to each test vessel

** Three fish observed to be swimming normally, but exhibiting mild effects (e.g. mild hyperventilation, mild pigment changes, swimming position in test vessel different to controls)

*** After approximately 2, 2.5, 3 and 4 hours exposure, a single fish, two fish, two fish and the remaining fish, respectively, were removed and euthanised due to severity limit

**** After approximately 22 and 24 hours exposure, a single fish and two fish, respectively, were removed and euthanised due to severity limit

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the initial 0-hour measured concentrations, the 96-hour LC50 was determined to be 18 mg/L and the No Observed Effect Concentration (NOEC) was determined to be 12 mg/L. The lowest concentration at which 100% mortality occurred was 38 mg/L.

Executive summary:

The study was conducted in accordance with OECD Guideline No. 203, under GLP conditions to assess the toxicity the 96-hour acute toxicity of the substance to rainbow trout (Oncorhynchus mykiss). The definitive test was conducted at nominal test concentrations of 10, 18, 32, 56 and 100 mg/L under static conditions at a single nominal loading rate of 100 mg/L with a concurrent control group, each consisting of a single 20 L constructed glass aquaria containing 15 L of media. Following acclimation to the test conditions for at least 12 days, seven fish were added to each test vessel. TheDaphnia magnain each test vessel were observed for mortality and sub-lethal effects at ca. 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The fish were not fed during the test.  

Test solutions were analysed at 0 and 96 hours using high performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of phosphoric acid dimethyl ester constituent, the marker constituent with the most singularly abundant peak.Effect concentrations were based on the initial 0-hour measured concentrations:  7.1, 12, 22, 38 and 71 mg/L.

The 96-hour LC50was determined to be 18 mg/L and the No Observed Effect Concentration (NOEC) was determined to be 12mg/L.The lowest concentration at which 100% mortality occurred was 38 mg/L.

Description of key information

In an OECD Guideline No. 203 study, conducted according to GLP, to assess the toxicity of the substance to the freshwater fish species, Oncorhynchus mykiss, the 96-hour LC50 was determined under static test conditions to be18 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 12 mg/L, based on initial measured concentrations (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

18 mg/L