Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

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:

from 2018-10-05 to 2018-10-12 with a definitive exposure phase from 2018-10-08 to 2018-10-12 at the test facility

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide

Trade Name Hostacor ITD

EC no. 947-922-8

Batch no. DEG4 410913

Purity
(certified) 98.1%

Constituent Constituent:
C11-15 Isoalkyl dihydrogen phosphate 36.2% (w/w)
C11-15 Diisoalkyl hydrogen phosphate 55.9% (w/w)
C11-15 Triisoalkyl phosphate 0.5% (w/w)
Pyroester 5.5% (w/w)

By-products:
C11-15 Isoalkyl alcohol 0.7% (w/w)
Phosphoric acid 1.2% (w/w)
Water 0.1% (w/w)

Water solubility 1 mg/L

Density approx. 0.98 g/cm3 (20 °C)

Appearance Clear yellowish liquid

Expiry date 2019-03-31

Recommended storage Keep container tightly closed in a cool (+2 - +8 °C), dry and well-ventilated place

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The saturated solution and the control were analytically verified via LC-MS/MS in the fresh media at the start of exposure and at renewal (0 and 72 hours) and in the 24-hours old media after 24 and 96 hours.The analytical method validation was done according SANCO/3029/99 rev.4 (2000) which is consistent with the ECHA guidance on information requirements (Version 1, July 2013).

Sampling for the analytical monitoring
At the start of the exposure and at renewal (0 and 72 hours), samples of the fresh media were taken after preparation of the saturated solution and analyzed.
At renewal and at the end of the exposure (24 and 96 hours), samples of the 24-hours old media were taken from the test vessels for analyses.

Vehicle:

no

Details on test solutions:

Test method
A semi-static test with daily renewal of the test media was performed.

Limit concentration
A saturated solution prepared with a nominal concentration of 100 mg/L of the test item was tested. Taking into account the aquatic toxicity data of the test item, the test was performed without a range-finding test. The threshold concentration (TC) was based on a Daphnia toxicity test of the test item:
EC50 > saturated solution (nominal loading 100 mg/L), HERRMANN, 2018 and an algae toxicity test of the test item (calculated test item concentration of the saturated solution (nominal loading 100 mg/L)): ErC50 (0-96 h) 1.90 mg/L and EyC50 (0-96 h) 0.963 mg/L, HERRMANN, 2018.

Preparation of the saturated solution
A saturated solution of 100 mg test item/L was prepared in dilution water three days before the start of the exposure (at day -3) and three days before each renewal of the test solutions (at day -2, -1 and 0).
For each preparation day two glass bottles were filled each with 2 L of the dilution water, an appropriate volume of the test item (density: 0.98 g/cm³ (20 °C) was taken into account) was placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 72 hours with a magnetic stirrer at room temperature.
 
After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel).
The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated test item solution, was used as limit concentration in the test. During filtration, the filter was always kept covered.

Control 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.54 cm
- Weight at study end (mean and range, SD): Average body weight: 0.117 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (7 - 750 lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within the last 7 days before the start of the exposure were used in the test. No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.

- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 73 mg CaCO3/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on results incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume:
Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates.
Test volume: About 2.5 L per vessel

- Aeration: Gentle aeration was provided
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.328 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerate for at least 24 h to remove chlorine.

Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.10 mmol/L (recent measurement 2018-08-28)
Alcalinity: 0.70 mmol/L (recent measurement 2018-08-28)
Conductivity: 144 µS/cm (recent measurement 2018-08-28)

- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were measured in all test vessels at the start and end of the
exposure and every 24 hours. Total hardness was determined at the start of the exposure out of the control. During the test the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 3, 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.585 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: See tables below
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solution was visible clear throughout the period of exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation
The LC0 after 96 h was empirically derived from the raw data of the limit concentration. LC50-values do not have to be calculated in a limit test.

Software
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

The following software was used for calculations:
Excel, MICROSOFT CORPORATION

Sublethal observations / clinical signs:

Preliminary Test (non-GLP)

 

A non-GLP stability test without fish under static conditions over a period of 96 hours was conducted at the test facility with the nominal concentration of 100% of the saturated solution.The saturated solution was prepared and had a nominal loading of 100 mg/L. Tyndall effect was negative in the freshly prepared saturated solution and the test medium wasvisually clear throughout the test period. Sampling for analytical determination of the test item was carried after 0, 48 and 96 hours of exposure.The measured concentrations of the saturated solution are given in the table below.

 

Measured Exposure Concentrations during the non-GLP Preliminary Range Finding Test

Determination via LC-MS/MS

Sampling	0 hours	48 hours		96 hours
Saturated solution [%]	Reactionproducts of alcohols,C11-14-iso,C13 rich and phosphorus pentoxide
	Measured concentration [mg/L]	Measured concentration [mg/L]	%	Measured concentration [mg/L]	%
100	0.367	0.261	72	0.312	86

% = percent of the initially measured test item concentration

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels

                  (n = 7, number of fish)

 

Geometric mean measured test item concentration [mg/L]		Effect *		Number of fish effected at observation time [hours]
			3		24		48		72		96
0.585		(1)		7		7		7		7		7
Control		(1)		7		7		7		7		7

              *) The numbers in brackets correspond to the following observations: 

                         (1)      =Normal behavior

 

 

Cumulative Mortality[%]in the Test Vessels

Geometric mean measured test item concentration [mg/L]	Cumulative mortality at observation time [hours]
	3	24	48	72	96
0.585	0	0	0	0	0
Control	0	0	0	0	0

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item Reaction products ofalcohols, C11-14-iso,C13 rich and phosphorus pentoxidewere analytically verified via LC-MS/MS from freshly prepared media after 0 and 72 hours and from the corresponding 24 hours aged test media after 24 and 96 hours in the limit tested concentration and the control.

 

The mean measured concentrations at the start of the exposures were 1.61 mg/L (0 h) and 0.451 mg/L (72 h). At the end of exposures, the measured concentrations were 33% (24 h) and 66% (96 h) of the initially measured test item concentrations. All solutions were prepared from the saturated solution and the dilution factor was reflected in the measured concentrations. The exposure concentrations were calculated from the value for the saturated solution.All effect values given are based on the geomean test item concentrations of Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide.

 

 

Measured Concentrations and Percent of the Nominal Concentrations of the Test Item

Sampling date	2018-10-08 0 hours (Fresh medium)	2018-10-09 24 hours (Old medium)		2018-10-11 72 hours (Fresh medium)	2018-10-12 96 hours (Old medium)
Start of analysis	2018-10-08	2018-10-09		2018-10-11	2018-10-12
Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide
Dilution level of the saturated solution [%]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Geometric mean meas. concentration [mg/L]
100	1.61	0.539	33	0.451	0.300	66	0.585
Control	< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc.    =Measured concentration of the test item, dilution factor taken into account, mean value of two injections

%                     =Percent of initial concentration of the test item

LOQ                         = Limit of Quantification (0.3 mg/L test item)

 

 

Since during the test no mortalities occurred, no regression analysis for the concentration-effect relationships was carried out.

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the geometric mean measured threshold concentration of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide. The threshold concentration of the test item at the level of water solubility was found to have no effects on zebrafish.
In conclusion, the LC0 corresponds to the geometric mean measured concentration of 0.585 mg test item/L.

Executive summary:

The acute toxicity of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide to fish (zebrafish) was determined according to the principlesof the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from2018-10-05 to 2018-10-12 with a definitive exposure phase from 2018-10-08 to 2018-10-12 at the test facility.

 

An acute toxicity test with thesaturated solution with a nominal loading of 100 mg/Lwas conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the saturated solution and the control, each. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

 

The saturated solution of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide was analytically verified via LC-MS/MS from freshly prepared test media after 0 and 72 h and from the corresponding 24 h aged test media after 24 and 96 h. The measured concentrations of the saturated solution in the fresh media (0 and 72 h) were 1.61 and 0.451 mg/L. The measured concentrations in the old media (24 and 96 h) were 33 and 66% of the initially measured concentrations, respectively. Therefore, all effect values were based on the geometric mean measured concentration of the test item.

 

All validity criteria of the test guideline were met .

 

LL/LC-Values(3 – 96 hours) of the Test Item

Based on the geometric mean measured test item concentration and nominal test item loading [mg/L]

 

	Geometric mean measured test item concentration	Nominal test item loading
LL/LC0= Highest test item loading/concentration with 0% mortality after 96 h	0.585 mg/L	100 mg/L

 

Description of key information

All effect levels are given based on the geometric mean measured threshold concentration of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide. The threshold concentration of the test item at the level of water solubility was found to have no effects on zebrafish.

In conclusion, the LC0 corresponds to the geometric mean measured concentration of 0.585 mg test item/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L