Ammonium (xylenes and 4-ethylbenzene)sulfonates

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from another member of the category

Adequacy of study:

key study

Study period:

April 21 - 25, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without GLP or analytical confirmation

Justification for type of information:

Please see attached Category Approach

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 797.1400 (Fish Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

no

Remarks:

as stated in protocol

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: Oncorynchus mykiss
- Source: SLI Lot #93A5
- Age at study initiation (mean and range, SD): not mentioned
- Length at study initiation: a representative sample (30) of fish from the test population has a mean lenght of 39 mm (range 32 to 45 mm)
- Weight at study initiation: a representative sample (30) of fish from the test population has a mean weight of 0.43 g (range 0.26 to 0.93 g)
- Method of breeding: the fish were fed a dry commercial pellet food, ad libitum, daily except the 48 hours prior to testing. Fish were not fed during the test.

ACCLIMATION
- Acclimation period: 14 day minimum
- Acclimation conditions (same as test or not): same
- Type and amount of food: dry commercial pelleted food, ad libitum
- Feeding frequency: daily- Health during acclimation (any mortality observed): not mentioned

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

36 mg/L

Test temperature:

12+/- 1 degree C

pH:

7.0 - 7.4

Dissolved oxygen:

78-92% of saturation

Nominal and measured concentrations:

0, 130, 220 360, 600 and 1000 mg active ingredient/liter (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass, 18.9 liter, 15 liter of test solution
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 0.29 grams per liter of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Total organic carbon: 0.82 - 1.5 mg/L
- Particulate matter: not mentioned
- Metals: below limits of quantitation except 0.02 mg/L manganese and 2.3 mg/L magnesium
- Pesticides: all less than limits of quantitation
- Alkalinity: 26 mg/L as CaC03- Ca/mg ratio: 3:1
- Conductivity: 100 micro mhos/cm
- Culture medium different from test medium: same
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
:- Photoperiod: 16 hrs light - 8 hrs darkness
- Light intensity: 70 footcandles at surface

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and observations of the fish twice daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: <2X
- Range finding study
- Test concentrations: 0.10 to 100 mg active ingredient per liter
Results used to determine the conditions for the definitive study: no mortality or sublethal effects 8e.g. lethargy, loss of equilibrium etc) were observed at any concentrations.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: no mortality observed in any test chambers

Details on results:

- Behavioural abnormalities: no loss of equilibrium observed
- Mortality of control: none
- Other adverse effects control: none

Results with reference substance (positive control):

no reference substance used

Validity criteria fulfilled:

yes

Conclusions:

LC50 = 1000 mg/L active ingredient

Executive summary:

The acute toxicity of the Sodium (Xylene and 4-ethylbenzene) sulfonate to fish (Oncorynchus mykiss) was assessed according to the principles of the EPA OTS 797.1400. The study was performed over a period of 96 hours under static conditions. Ten test organisms were exposed to the test concentration and the control, respectively. 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 test solutions were visually clear throughout the study. All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the highest test concentration of 1000 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from another member of the category

Adequacy of study:

supporting study

Study period:

July 15 - 19, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP compliance; full documentation

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1400 (Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: stock solution of 2,050 mg/L and mean measured concentrations of 114, 227, 428, 824 and 1580 mg/L plus a control. All test concentrations were corrected for the 31.2% sample activity.
- Sampling method: samples collected at 0 and 96 hours of the test. Measurements using HPLC procedure.
- Sample storage conditions before analysis: not specified

Vehicle:

no

Details on test solutions:

A proportional diluter and syringe dispenser system delivered the test material. Nominal concentrations were 112, 224, 448, 895 and 1790 mg/L. The material was dissolved in hard water. All solutions were clear with no visible precipitate. The flow rate was equivalent to 5.9 tank volume replacements daily. The test solution temperature was maintained at 12 +/- 1 degree C.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Mt Lassen Trout Farm in Red Bluff, California
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 5.4 +/- 0.3 cm
- Weight at study initiation (mean and range, SD): 2.24 +/- 0.41 g
- Method of breeding: fertilization of eggs with milt at the laboratory
- Feeding during test: not fed

ACCLIMATION
- Acclimation period: 72 hours
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: not fed
- Health during acclimation (any mortality observed): no data

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

150-154 mg/L

Test temperature:

11-13 degrees C

pH:

7.7 - 8.3

Dissolved oxygen:

7.6 - 8.8 mg/L (equivalent to 74 and 87% saturation)

Nominal and measured concentrations:

112, 224, 448, 895 and 1790 mg/L nominal
113, 227, 428, 824 and 1580 mg/L measured

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 15 L glass aquaria filled to 12 L
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 71 L of water per day per aquarium
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): no vehicle
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory hard blended water
- Total organic carbon: <1.0 mg/L
- Particulate matter: 0.3 mg/L
- Metals, Pesticides: checked
- Alkalinity: 158-160 mg/L
- Conductivity: 320 umhos/cm
- Culture medium different from test medium: same
- Intervals of water quality measurement: 0, 24, 48, 72 and 96 hours

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours of light
- Light intensity: 625-654 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and sublethal effects at each 24 hour interval

TEST CONCENTRATIONS
- Spacing factor for test concentrations: proportional diluter
- Range finding study used to establish test concentrations
Static range-finding tests were conducted to determine the concentration range to use in the definitive test. The test concentrations during the first test were set at 1, 10, and 100 mg/L. The second test was conducted at the test concentrations of 1000 and 10000 mg/L. Five fish were exposed to each test concentration in these tests. The 10000 mgIL concentration resulted in 100 % mortality within 24 hours. No mortality nor abnormal effects were noted in any other concentration during the 96 hour tests. All solutions were clear with no visible precipitate. The 10000-mg/L solution had a light surface scum visible at 0 hour which was not visible after this time.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 824 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 1 580 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: fish on bottom of test chamber, loss of equilibrium, surfacing, dark discoloration, quiescent and labored respiration were observed in some or all of the fish in both replicates at the high exposure (1580 mg/L). No sublethal effects were observed at any other exposure.
- Mortality of control: none
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no precipitation observed

Validity criteria fulfilled:

yes

Conclusions:

LC50 >1580 mg/L 96-hr, active ingredient
NOEC = 824 mg/L

Executive summary:

The acute toxicity of the test item Calcium xylenesulphonate to fish (Oncorynchus mykiss) was assessed according to the principles of the EPA OTS 797.1400 (Fish acute toxicity test). The study was performed over a period of 96 hours under flow-through conditions. ten test organisms were exposed to the test concentration and the control, respectively. 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 test solutions were visually clear throughout the study. The concentration of the test item Calcium xylenesulphonate in aquatic test water was analytically verified via HPLC in fresh media at the start of the exposure (0 hours and 96 hours). Control and tested item samples were determined at each sampling period.
All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the test concentration 1580 mg/L.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from another member of the category

Adequacy of study:

supporting study

Study period:

2020

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)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A limit concentration of 252 mg/L corresponding to 100 mg a.s./L, was tested as a threshold concentration. The selection of the test concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EC50 (0-48 h) = >304 mg test item /L, corresponding to 121 mg a.s./L) and of an alga toxicity test (ErC50 (0-72 h) = > 252 mg test item/L, corresponding to 100 mg a.s./L).
- Sampling method: Dilution medium fish dilution water : acetonitrile (75 : 25)
Preparation of the standards: A stock solution of the test item of 100 mg/L in ultra-pure water was diluted in two steps to 100 μg/L standard with dilution medium. Out of this solution, 5 further concentrations with dilution medium were prepared for a calibration range from 10 to 100 μg/L.
Preparation of the samples: The limit test item loading rate and the control were diluted with
acetonitrile (factor 1.3) directly after sampling.
- Sample storage conditions before analysis: Storage condition 18 – 25 °C, dark, in tightly closed original container

Vehicle:

yes

Details on test solutions:

Preparation of the test medium: An appropriate amount of the test item was weighed and transferred into a test vessel filled with an appropriate amount of the dilution water. The test media was mixed with an ultraturrax (1 min, 17000 rpm). This procedure was repeated for every water
renewal.
Test vessel: Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with a glass plate.
Test volume: About 2.5 L per vessel
Aeration: No aeration was provided.
Dilution water: Same as used for holding

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Origin: All fish used in the test were gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
Holding: Holding was performed in accordance with the laboratory SOPs at the test facility at 23 ± 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.
Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to 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.
Water: Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 40 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Oxygen saturation: ≥ 80 % of air saturation value
Acidity: 0.2 mmol/L (recent measurement 2020-04-27)
Alkalinity: 0.5 mmol/L (recent measurement 2020-04-27)
Conductivity: 144 μS/cm (recent measurement 2020-04-27)
Feeding: Food was provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. The test fish were not fed 24 h before test start.
Food: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

Observations were made after 2 ± 0.5 h, 5 ± 1 h and 24 ± 2 h after the start of the exposure. On days 2-4 of the test, all vessels with living fish were inspected twice per day.

Hardness:

Total hardness: 40 - 250 mg CaCO3/L

Test temperature:

21 - 25 °C, controlled at ± 1 °C

pH:

pH-value: 6.0 - 8.5

Dissolved oxygen:

Not less than 60% of air saturation value

Conductivity:

144 μS/cm

Nominal and measured concentrations:

252 mg test item /L corresponding to 100 mg a.s./L were tested as a threshold concentration in a limit test

Details on test conditions:

Control: 7 fish in dilution water (without test item but treated in the same way as the test solution) were tested under the same test conditions as the test replicates.
Test vessel: Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with a glass plate.
Test volume: About 2.5 L per vessel
Aeration: No aeration was provided.
Dilution water: Same as used for holding
Acclimatization: Acclimatization was not necessary, because the quality of the dilution water was equivalent to the water used for holding.
Number of test fish: 7 zebrafish were used each for the limit concentration and the control. For the whole study 14 fish were used.
Loading, length of fish : The fish density in the test vessels was 0.0868 g fish per liter test solution. Average body length at the test end: 1.73 cm
Average body weight at the test end: 0.031 g
Age of fish: Juvenile
Replicates: One replicate each for the limit concentration and the control
Introduction of fish: The fish were randomly distributed among the treatments.
Feeding of test fish: The fish were not fed during the exposure.
Water temperature (target): 21 - 25 °C, controlled at ± 1 °C
Dissolved oxygen Concentration (target): Not less than 60% of air saturation value
Light intensity (target): 540 – 1000 Lux
Photoperiod: A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 252 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The threshold concentration of the test item at the nominal concentration 252 mg/L corresponding to 100 mg a.s./L was found to have no lethal or toxic effects on zebrafish.

Sublethal observations / clinical signs:

The study was performed according to the OECD-Guideline No. 203 (2019) for Testing of Chemicals and GLP principles and met the validity criteria:
- The O2 saturation should be  60% (this study: ≥ 82%).
- The mortality in the control group should not exceed 1/7 fish (this study: 0/7 fish)
- Recovery rates of the test item 80 - 120% throughout the test.
The measured concentrations of the test item were 91% and 100% in the fresh media and 91% in the old media of the nominal test item concentration

 

Table 1:    LC₀-Value (96 hours) of the Test Item

   Based on the nominal concentrations of the test item and the active substance [mg/L]

 

LC0 = Highest test item concentration with 0% mortality after 96 h	Test item concentration	a.s. concentration
	≥ 252 mg/L	≥ 100 mg/L
LC50 = Highest test item concentration with 50% mortality after 96 h	> 252 mg/L	> 100 mg/L

 

 

Table 2:    Observations of Sub-Lethal Effects and Normal Behavior in the Test Vessels

                   (n = 7, number of fish)

 

Nominal Test item concentration [mg/L]	Effect *	Number of fish affected at observation time [hours]
		day 0		day 1	day 2		day 3		day 4
		2	4	24	45	52	69	75	92	96
252	(1)	7	7	7	7	7	7	7	7	7
Control	(1)	7	7	7	7	7	7	7	7	7

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

                          (1)       = Normal behavior

 

Table 3:    Cumulative Mortality [%] in the Test Vessels

Nominal Test item concentration [mg/L]	Cumulative mortality [%] at observation time [hours]
	day 0		day 1	day 2		day 3		day 4
	2	4	24	45	52	69	75	92	96
252	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0

 

 

Table 4:     Environmental Conditions

	Nominal Test item concentration [mg/L]	Oxygen Saturation [%]		pH Value		Temperature [°C]
		old	new	old	new	old	new
Day 0	252		100		7.79		22.0
	Control		100		7.85		22.0
Day 1	252	83	98	7.39	7.74	22.1	21.6
	Control	85	92	7.36	7.41	22.2	21.7
Day 2	252	85	93	7.58	7.84	21.8	21.7
	Control	84	92	7.53	7.58	22.2	21.9
Day 3	252	84	92	7.59	7.61	22.1	21.6
	Control	83	90	7.52	7.56	22.0	21.6
Day 4	252	82		7.66		21.9
	Control	82		7.70		22.0

 

 

Table 5:    Water Temperature in the Control (Continuous Measuring)

 

Period of Measurements	2020-05-11 to 2020-05-15
Minimum Temperature [°C]	21.8
Maximum Temperature [°C]	22.2
Mean Temperature [°C]	21.9

 

Table 6:       Measured Concentrations of the Test Item sodium p-cumenesulphonate during the Definitive Test

Sampling date:	0 hours, Fresh medium		24 hours, Old medium
Nominal concentration of the test item [mg/L]	sodium p-cumenesulphonate
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
252	230	91	230	91
QC	29.9	101	29.4	99
Control	< LOQ		< LOQ
Sampling date:	72 hours, Fresh medium		96 hours, Old medium
Nominal concentration of the test item [mg/L]	sodium p-cumenesulphonate
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
252	253	100	228	91
QC	29.1	98	29.1	98
Control	< LOQ		< LOQ

 

Validity criteria fulfilled:

yes

Conclusions:

LC50 > 252 mg/L corresponding to 100 mg/l active ingredient

Executive summary:

The acute toxicity of sodium p-cumenesulphonate to fish (zebrafish) was assessed according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019).
A limit concentration of 252 mg/L corresponding to 100 mg a.s./L, was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the results of a daphnia acute immobilization test (EC50 (0-48 h) = > 304 mg/L, corresponding to 121 mg a.s./L, and an alga toxicity test (ErC50 (0-72 h) = > 252 mg/L, corresponding to 100 mg a.s./L (both nominal test item concentration)).
The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. Seven test organisms were exposed to the test concentration and the control, respectively. 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 test solutions were visually clear throughout the study.
The concentration of the test item sodium p-cumenesulphonate was analytically verified via LCMS/ MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours) in the limit test item concentration and the control. The measured concentration of the test item were 91% and 100% in the fresh media and 91% in the old media of the nominal test item concentration.
All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the test concentration 252 mg/L, corresponding to 100 mg a.s./L.

Description of key information

The HYDROTOPES Category comprises the following 6 substances:
STS - Sodium toluene 4-sulphonate (CAS 657-84-1, EC 211-522-5)
SXS - Sodium (xylenes and 4-ethylbenzene) sulphonate (EC 701-037-1)
NH4XS - Ammonium (xylenes and 4-ethylbenzene) sulphonate (EC 943-024-5)
SCS - Sodium p-cumenesulphonate (CAS 15763-76-5, EC 239-854-6)
KCS - Potassium p-cumenesulphonate (CAS 164524-02-1, EC 629-764-9)
NH4CS - Ammonium p-cumenesulphonate (CAS 680972-33-2, EC 811-484-5) 
In addition CaXS (Calcium Xylenesulphonate, CAS 28088-63-3, EC 248-829-9) was evaluated for complete the assessment despite it is not registered under REACH.

There are available studies on short term toxicity to fish performed on two subgroups (xylene and cumene) and the missing toluene subgroup is covered by the data on the related sulphonic acid. The results of the available studies are:
SXS (1993, no GLP): LC50 > 1000 mg/l (a.i.)
CaXS (1994, GLP): LC50 > 1580 mg/l (a.i.)
SCS (2020, GLP): LC50 > 100 mg/l (a.i.)
TSA (1981, no GLP): LC50 > 325 mg/l (a.i.)

The key study used for the assessment is the test performed on SCS (2020, GLP) with the 96-hour LC50 value > 100 mg/L.
The other available studies are:
- a 1993 USEPA Guideline study (EPA-OTS 5797.1400) Fish Acute Toxicity Test using Oncorhynchus mykiss with the test substance sodium xylenesulphonate. The study is well documented but was not conducted under GLP compliance. Rainbow trout were exposed to the test substance for 96 hours in freshwater under static conditions. Chemical analysis was not performed. The nominal 96-hour LC50 was greater than the highest test concentration at >1000 mg/L (active ingredient). No deaths or abnormal behavior were observed in the test at any of the 5 concentrations.
- a USEPA Guideline (EPA-OTS 5797.1400) with the tested substance calcium xylenesulphonate and conducted to GLP compliance. The measured 96-hour LC50 for rainbow trout in this flow-through study was 1580 mg/L. The NOEC was 824 mg/L based on behavioural abnormalities (e.g., loss of equilibrium) at the 1580 mg/L exposure level.
These studies indicate that of the Hydrotropes substances are comparable in term of effects on fish and show low toxicity.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

100 mg/L