A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2014 - 11 July 2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 2: Semi-static method)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples
The test samples were stored in the freezer (≤ -15°C).
On the day of analysis, the test samples were defrosted at room temperature. The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 50/50 (v/v) acetonintrile/ISO-medium to obtain concentrations within the calibration range.

Vehicle:

yes

Details on test solutions:

The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturbed the test system was prevented (e.g. film of the test substance on the water surface).

The batch of ANOX® 1315 tested was a clear yellow viscous liquid with a purity of 93.5% by GC and not completely soluble in test medium at the loading rates initially prepared.
Preparation of test solutions started with individual loading rates of 1.0, 10 and 100 mg/l. A two-day period of magnetic stirring was applied and was followed by a one-hour settlement period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off and used as test solutions. The final test solutions were all clear and colourless.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758

Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.

Mean length*: Range-finding test: 2.9 ± 0.5 cm. Final test: 1.8 ± 0.28 cm

Mean weight*: Range-finding test: 0.64 ± 0.36 g. Final test: 4.0 ± 0.2 g

Characteristics: F1 from a single parent-pair bred in UV-treated water.

Reason for selection: This system has been selected as an internationally accepted species.

Total fish used: 23

*Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Quarantine/Acclimatisation: At least 12 days after delivery.

Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l

Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week.

Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.

Water quality parameters: Were kept within the optimum limits for the respective fish species (pH between 6.0 and 8.5, nitrate <200 mg/l, nitrite <0.10 mg/l and ammonia <0.5 mg/l).

Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)

Validity of batch of carp: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

180 mg CaCO3 per litre

Test temperature:

20-24°C

pH:

6.0-8.5

Dissolved oxygen:

> 60% of air saturation

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations of 1.0, 10 and 100 mg/l

Details on test conditions:

Test duration: 96 hours

Test type: Semi-static, with daily renewal of test solutions

Test vessels: 25 litres, all-glass, containing 25 litres of test solution

Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.

Number of fish: 7 fish per concentration and control

Loading: 0.5 g fish/litre, i.e. 7 fish per 25 litres of test medium

Illumination: 16 hours photoperiod daily (05:00-21:00)

Aeration: The test media were not aerated during the test.

Feeding: No feeding from 24 hours prior to the test and during the total test period.

Introduction of fish: Within 0.5 hour after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5°C.

Euthanasia: At the end of the test the surviving fish of the limit concentration were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water. Fish exposed to the control group were instantly killed by a blow on the back of the head followed by cervical incision.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 0.19 mg/l being considered the maximum solubility limit in test medium.

Details on results:

Experimental conditions
All test conditions remained within the ranges prescribed by the protocol (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Range-finding test
No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from the WAF prepared at 100 mg/l were analysed. The initial concentration was 14 mg/l and decreased to 0.63 mg/l (i.e. 4.6% of initial) at the end of the test. It should be noted that a floating layer of test substance was observed in the highest concentration already after 2½ h of exposure and in the WAF prepared at 10 mg/l after 24 hours of exposure, indicating that these two solutions were oversaturated. Therefore, it was decided, in consultation with the sponsor, to perform the final test semi-statically with WAF prepared at 1.0 mg/l as a limit concentration.
All test conditions were maintained within the limits prescribed by the protocol.

Limit test
Measured concentrations
The actual concentration measured in the limit concentration was 0.17 and 0.25 mg/l in the freshly prepared solutions (i.e. t=0 and 72h, respectively). The measured concentration remained stable during the refreshment period (81-85% of initial). The calculated average exposure concentration corresponded to 0.19 mg/l.

Mortality and other effects
The responses recorded in this test allowed for reliable determination of an LC50. The responses recorded at the limit concentration were in agreement with the results of the range-finding test. No mortality or other clinical effects were observed in this test.


Analytical results

Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

Procedural recovery samples

The chromatograms of the blank procedural recovery samples showed no peak at the retention time of the test substance. It demonstrated that the sample treatment was adequate for the test samples.

The mean recoveries of the procedural recovery samples containing test substance fell within the criterion of 70 − 110%. It demonstrated that the analytical method was adequate for the determination of the test substance concentration in the test samples.

Results with reference substance (positive control):

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.

Under the conditions of the present test PCP induced no lethal effects in carp at 0.10 mg/l. The
96h-LC50 for carp exposed to PCP was 0.24 mg/l. This effect was already reached within 48 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the range-finding test

ANOX® 1315 WAF prepared at (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2½h	24h	48h	72h	96h
1	3	0	0	0	0	0	0
10	3	0	0	0	0	0	0
100	3	0	0	0	0	0	0

 

Incidence of mortality and total mortality during the limit test

Average exposure concentration ANOX® 1315 (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2½h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
0.19	7	0	0	0	0	0	0

 

Determination of effect concentrations

Parameter	Average exposure concentration ANOX® 1315 (mg/l)
4, 24, 48, 72 and 96h-LC50	>0.19

 

pH-values during the limit test

Average exposure concentration ANOX® 1315 (mg/l)	Day 0*		Day 1		Day 2		Day 3	Day 4
	Fresh	Old	Fresh	Old	Fresh	Old	Fresh	Old
Control	7.6	7.4	7.6	7.4	7.5	7.4	7.5	7.5
0.19	7.9	7.6	8.0	7.5	8.0	7.6	8.1	7.6

* pH of culture medium was: 8.0

Dissolved oxygen concentrations during the limit test

Average exposure concentration ANOX® 1315 (mg/l)	Day 0		Day 1		Day 2		Day 3	Day 4
	Fresh	Old	Fresh	Old	Fresh	Old	Fresh	Old
Control	8.8	6.8	9.1	6.9	9.0	6.6	8.9	6.6
0.19	8.8	7.3	9.1	7.2	9.0	7.1	8.9	7.2

 

Temperatures (°C) measured during the limit test

Average exposure concentration ANOX® 1315 (mg/l)	Day 0*		Day 1		Day 2		Day 3	Day 4
	Fresh	Old	Fresh	Old	Fresh	Old	Fresh	Old
Control	22	22	22	22	22	22	22	22
0.19	22	22	22	22	22	22	22	22

*Temperature of culture medium was: 23˚C

 

Incidence of mortality observed in the reference study:

Concentration PCP (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2½h	24h	48h	72h	96h
0.10	5	0	0	0	0	0	0
0.22	5	0	0	1	1	1	20
0.46	5	0	5	5	5	5	100

 

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 exceeded an average exposure concentration of 0.19 mg/l being considered the maximum solubility limit in test medium.

Executive summary:

96-Hour Acute Toxicity Study in Carp with ANOX® 1315.

 

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008, the ISO International Standard 7346, 1996 and the OECD series on testing and assessment number 23, 2000.

 

The batch of ANOX® 1315 tested was a clear yellow viscous liquid with a purity of 93.5% by GC and not completely soluble in test medium at the loading rates initially prepared.

 

A limit test was performed based on the results of a range-finding test. A Water Accommodated Fraction (WAF) was prepared at a loading rate of 1.0 mg/l. A two-day period of magnetic stirring was applied and was followed by a one-hour settlement period. Thereafter, the aqueous WAF was siphoned off and used as test solution. The final test solutions were all clear and colourless.

Seven fish per group were exposed to a control and a WAF prepared at a loading rate of 1.0 mg/l in the limit test. The total exposure period was 96 hours and the solutions were refreshed daily. Samples for analytical confirmation of the exposure concentrations were taken at the start and at the end of the first and the last refreshment period.

 

The actual concentration measured in the limit concentration was 0.17 and 0.25 mg/l in the freshly prepared solutions. The measured concentrations remained stable during the refreshment period (81-85% of initial). The calculated average exposure concentration corresponded to 0.19 mg/l.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

The 96h-LC50 exceeded an average exposure concentration of 0.19 mg/l being considered the maximum solubility limit in test medium.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 September 1992 - September 25 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

Preparation: solutions were prepared by dissolving weighed amounts of test article directly in the treatment tanks in order to obtain the requested concentration of 0.33 mg/l

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Brachydanio rerio (Hamilton Buchanan).

Justification for the selection of the test system: Brachydanio rerio was chosen as a Cyprinidae species, since it is wideIy accepted by Health Authorities as an experimental model with documented susceptibility to a wide range of toxic substances.

Number: 10 animaIs/concentration

Body weight and length: 0.384 g and 3 cm (average values).

Supplier: "AQUA", Piazza Pistoni 10, 10015 IVREA (Torino, Italy).

Acclimation: about 20 days before start of test. All fish were exposed (in a glass aquarium) to water having the characteristics listed below.

Water characteristics:
- Source: from the municipal water main system
- Temperature: 22˚ C
- pH: 7.2
- Hardness: 55 mg/l of CaCO3
- O2 concentration: 8.9 mg/l of the air saturation volume

These characteristics were measured immediately before the arrival of the fish.
Periodically, the water was analyzed for microbiologic count, heavy metals and other chemical and physical characteristics.

The acceptance limits of quality of the water were those defined in EEC Directive 801778.

Feeding: daiIy with Mangime SERA DISKUS - Aquaristik GmbH, manufactured on April 1992 (stable at least 15 months) until 24 hours before the test starts.
Contaminants that might interfere with the objectives of the study were not expected to be present either in the feed or in the water.

Mortality: following a 43-hour settling in period, mortality was recorded as 0% of the population after 7 days of acclimation.

Light: 12-16 hours of photoperiod daily.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96 hours. treatment solutions were changed every 24 hours (Semi-static test).

Hardness:

55 mg CaCO3/L

Test temperature:

22˚C

pH:

7.2

Dissolved oxygen:

O2 concentration: 8.9 mg/l of the air saturation volume

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

0.33 mg/l (Nominal)

Details on test conditions:

Experimental design: on request of the Sponsor, the test article was tested at the concentration of 0.33 mg/l that is the maximum solubility of the test article in water. One group of 10 animals was created only with water (control group).

Housing of the experimental groups: 10 animals/makrolon tank containing 10 liters of aerated
tap water. Aeration was done by means of compressed air.
Tank size: 48x27~20c m
Tank cards gave experiment number, nominal concentration and date of the treatment.
Treatment period: 96 hours. treatment solutions were changed every 24 hours (Semi-static test).

Loading: maximum loading was lower than 1.0 g fish/l (about 0.384 g/l)

Observation period: 96 hours

Observation of mortality: the fish were inspected after 24, 48, 72 and 96 hours. Fish were considered dead if touching of the caudai peduncle produced no reactions.

Observation of visible abnormalities: records were kept of effects other than lethal; these include all effects observed in the appearance, size and behaviour of the fish that make them clearly distinguishable from the control animals, e.g. different swimming behaviour, changes in appearance (discoloration).

Parameter checking:
- oxygen and temperature in test and control solutions were measured daily by means of a oxymeter YIS model 54 RL.
- pH of the test and control solutions was measured daily by means of a pHmeter
Radiometer PHM8O standard.

LC50 and its statistical limits: was not calculable.

Reference substance (positive control):

not specified

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Observation of visible abnormalities: No animals in the treated and in control group showed visible abnormalities at any observations.

Mortality: No animals in the treated or in the control group died.

Parameter checking: The values of dissolved oxygen, pH and temperature always remained between the acceptable limits.

LC50 and its statistical limits: LC50 at 23,48,72 and 96 were not calculable.

Validity criteria fulfilled:

yes

Conclusions:

Experimental data from an acute toxicity study in which fish were treated wit the test article ANOX BF at the nominal concentration of 0.33 mg/l (maximum solubility in water). The animals were treated and observed for 96 hours.
No animals in the treated nor in the control group died or showed visible abnormalities.
The LC50 at 24, 48, 72 and 96 hours were not calculable.

Description of key information

Danio rerio (Brachydanio rerio)

The 96h-LC50 exceeded an average exposure concentration of 0.33 mg/l being considered the maximum solubility limit in test medium.

Cyprinus carpio

The 96h-LC50 exceeded an average exposure concentration of 0.19 mg/l being considered the maximum solubility limit in test medium.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Danio rerio (Brachydanio rerio)

Experimental data from an acute toxicity study in which fish were treated with the test article ANOX BF at the nominal concentration of 0.33 mg/l (maximum solubility in water). The animals were treated and observed for 96 hours.
No animals in the treated nor in the control group died or showed visible abnormalities.
The LC50 at 24, 48, 72 and 96 hours were not calculable.

 

Cyprinus carpio

96-Hour Acute Toxicity Study in Carp with ANOX® 1315.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008, the ISO International Standard 7346, 1996 and the OECD series on testing and assessment number 23, 2000.

The batch of ANOX® 1315 tested was a clear yellow viscous liquid with a purity of 93.5% by GC and not completely soluble in test medium at the loading rates initially prepared.

A limit test was performed based on the results of a range-finding test. A Water Accommodated Fraction (WAF) was prepared at a loading rate of 1.0 mg/l. A two-day period of magnetic stirring was applied and was followed by a one-hour settlement period. Thereafter, the aqueous WAF was siphoned off and used as test solution. The final test solutions were all clear and colourless.

Seven fish per group were exposed to a control and a WAF prepared at a loading rate of 1.0 mg/l in the limit test. The total exposure period was 96 hours and the solutions were refreshed daily. Samples for analytical confirmation of the exposure concentrations were taken at the start and at the end of the first and the last refreshment period.

The actual concentration measured in the limit concentration was 0.17 and 0.25 mg/l in the freshly prepared solutions. The measured concentrations remained stable during the refreshment period (81-85% of initial). The calculated average exposure concentration corresponded to 0.19 mg/l.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

The 96h-LC50 exceeded an average exposure concentration of 0.19 mg/l being considered the maximum solubility limit in test medium.