(xylenes and 4-ethylbenzene)sulfonic acids

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 analogue substance

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

no chemical analysis; Goldorfen is not the standard test species but is among those often tested

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

diluted in water only

Test organisms (species):

Leuciscus idus melanotus

Details on test organisms:

TEST ORGANISM
- Common name: Goldorfen
- Source: Paul Eggers, 2345 Hohenwestedt
- Age at study initiation (mean and range, SD): not indicated
- Length at study initiation (length definition, mean, range and SD): 5.5 - 6.6 cm
- Weight at study initiation (mean and range, SD): 1.5-2.7 g
- Feeding during test: no

ACCLIMATION
- Acclimation period: no indicted
- Acclimation conditions: same as test
- Type and amount of food: Tetra Min
- Feeding frequency: not indicated
- Health during acclimation (any mortality observed): not indicated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

114 mg CaCO3/L

Test temperature:

20 +/- 1 C

pH:

7.5 - 8.2 (except at 500 mg/L where it was 5.7)

Dissolved oxygen:

8.5 - 9.2 mg/L

Salinity:

N/A

Nominal and measured concentrations:

Nominal - 0, 10, 100 500 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium with 20L solution
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 20L
- Aeration: yes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: not indicated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Ca/mg ratio: 0.7
- Na/K ratio: 21
- Intervals of water quality measurement: 0, 2, 24, 48, 72 and 96 hours

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12/12
- Light intensity: 700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality daily; observations regularly

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5-10X
- Range finding study: not indicated

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

>325 mg/L based on active ingredient

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no difference compared to control group
- Observations on body length and weight: not indicated
- Other biological observations: macroscopic examination showed no changes
- Mortality of control: none

Validity criteria fulfilled:

yes

Conclusions:

LC50 > 500 mg/L 96 h corresponding to 325 mg/L a.i.

Executive summary:

The acute toxicity of Toluene 4-sulphonic acid to fish was assessed according official guideline OECD 203. Test substance was not toxic at 500 mg/L (325 mg/L active substance), the highest concentration tested.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from a member of the Hydrotropes 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 3

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from a member of the Hydrotropes 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.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

other: Read Across from analogue substance

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: 128 mg/L, corresponding to 84.4 mg active substance/L, was tested as a threshold concentration in a limit test. The selection of the test concentration was based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (ErC50 (0-
72 h) = 128 mg/L (nominal test item test item concentration)) and a daphnia acute immobilization test (EC50 (0-48 h) = > 152 mg/L (nominal test item concentration)).

- 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 to 1000 μg/L with dilution medium and further diluted to a 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 All samples were diluted with Acetonitrile (factor 1 1/3) directly after
sampling. Further dilutions were made prior to analysis with dilution medium.

- Sample storage conditions before analysis:All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis

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.
The pH was adjusted daily in the test solutions to 7.0 ± 0.2 with NaOH (1 M).

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

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 2020-01-21)
Alkalinity: 0.6 mmol/L (recent measurement 2020-01-21)
Conductivity: 149 μS/cm (recent measurement 2020-01-21)

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.
Acclimatization: Acclimatization was not necessary, because the quality of the dilution water was equivalent to the water used for holding.
Food: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

58 [mg/L as CaCO3]

Test temperature:

Mean Temperature [°C]: 22.1

pH:

7.0 ± 0.2

Details on test conditions:

Preparation of 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.
The pH was adjusted daily in the test solutions to 7.0 ± 0.2 with NaOH (1 M).

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.
Reference item No reference item is recommended for this test according to the
guidelines.
Duration of the test 96 hours
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
Number of 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 0.0812 g fish per liter test solution. Average body length at the test end: 1.66 cm
Average body weight at the test end: 0.029 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.:

>= 128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The LC50 96h is 128 mg/L nominal concentration, corresponding to 84.4 mg active substance/L.
The concentration of the test item 4 - hydroxybenzenesulphonic acid was analytically verified via
LC-MS/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
loading rate and the control. The measured concentration of the test item were 98% and 105% in
the fresh media and 98% and 102% in the old media of the nominal test item loading rate.
None of the fish died and all keep a Normal behavior.

Validity criteria fulfilled:

yes

Conclusions:

LC50 ≥ 128 mg/l at 96 h corresponding to 84.4 mg/l a.i.
LC0 ≥ 128 mg/l at 96 h corresponding to 84.4 mg/l a.i.

Executive summary:

The acute toxicity of the test item 4 - hydroxybenzenesulphonic acid to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019). A limit concentration of 128 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (ErC50 (0-72 h) = 128 mg/L (nominal test item concentration)) and a daphnia acute immobilization test (EC50 (0-48 h) = > 152 mg/L (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 pH was adjusted to 7.0 ± 0.2 with NaOH (1 M) in the test concentrations daily before insertion of the fish. The concentration of the test item 4 - hydroxybenzenesulphonic acid was analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at one renewal interval (0 hours and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours in the limit concentration and the control. The measured concentration of the test item were 98% and 105% in the fresh media and 98% and 102% in the old media of the nominal test item loading rate. All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the test concentration 128 mg/L.

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

other: Read across from analogue substance

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: 70 mg a.i./L corresponding to a test item concentration of 98.7 mg/L were tested as a threshold concentration in a limit test.
The selection of the test concentration was based on the derivation of a threshold concentration (TC) from an algae toxicity test (ErC50 (0-72 h) = 70 mg a.i./L) and a daphnia toxicity
(EC50 (0-48 h) = 100 mg a.i./L) given by the sponsor and in the REACH dossier.
- Sampling method: An appropriate amount of the test item was transferred into a test vessel filled with an appropriate amount of the dilution water. For preparation the density was taken into account. The test media was mixed with an ultraturrax (1 min, 17000 rpm). This procedure was
repeated for every water renewal
- Sample storage conditions before analysis:18 - 25 °C, dark, in the tightly closed original container

Vehicle:

yes

Details on test solutions:

Test vessel: Glass aquaria of 13 L were used, filled with approximately 10 L of dilution water (dimensions: 25.5/18/28 cm, depth of water: approx. 20 cm) and covered with glass plates.
Test volume: About 10 L per vessel
Aeration: No aeration was provided.
Dilution water: Same as used for holding
Acclimatization: Acclimatization and the environmental conditions were not necessary, because the quality of the dilution water was equivalent to the water 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
Alkalinity: 0.5 mmol/L
Conductivity: 157 μS/cm
Acclimatization A sufficient number of fish were acclimatized to dilution water for at least 9 days prior to start of the exposure. The environmental
conditions were equivalent to the test conditions.
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

Number of 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 0.020 g fish per liter test solution. Average body length at the test end: 1.40 cm
Average body weight at the test end: 0.029 g
Age of fish: Juvenile
Replicates: One replicate each for the limit loading 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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

see tables attached

Test temperature:

see tables attached

pH:

see tables attached

Nominal and measured concentrations:

70 mg a.i./L corresponding to a test item concentration of 98.7 mg/L were tested as a threshold concentration in a limit test.

Details on test conditions:

Preparation of the test medium: An appropriate amount of the test item was transferred into a test vessel filled with an appropriate amount of the dilution water. For preparation the density was taken into account. The test media was mixed with an ultraturrax (1 min, 17000 rpm). This procedure was repeated for every water renewal.
Control: 7 fish in dilution water (without test item but from the same source and age as used for the test solution) were tested under the same test conditions as the test replicate.
Test vessel: Glass aquaria of 13 L were used, filled with approximately 10 L of dilution water (dimensions: 25.5/18/28 cm, depth of water: approx. 20 cm) and covered with glass plates.
Test volume: About 10 L per vessel
Aeration: No aeration was provided.
Dilution water: Same as used for holding
Acclimatization: Acclimatization and the environmental conditions were not necessary, because the quality of the dilution water was equivalent to the water used for holding.
Light intensity (target) 540 – 1000 Lux
Photoperiod: A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 98.7 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 98.7 mg/L corresponding to 70 mg a.i./L was found to have neither lethal nor sub lethal effects on zebrafish.
The concentration of the test item Benzenesulphonic acid was analytically verified via LC-MS/MS
analysis of fresh media at the start of the exposure intervals (0 hours and 48 hours) and in the
corresponding old media at the end of the exposure intervals (48 hours and 96 hours) in the limit
test item nominal concentration and the control.
The measured concentrations of the test item were 104% and 107% of the nominal concentration
in the fresh media at start of the exposure intervals and 100% and 101% at the end of the
exposure intervals.
The test solution was visibly clear throughout the period of exposure. The environmental
conditions (pH-value, temperature and oxygen-saturation) were determined to be within the
acceptable limits (see Table 4 and Table 5 attached). Light intensity was measured at the start of the exposure and ranged from 625 to 857 (mean value 725) Lux.

Validity criteria fulfilled:

yes

Conclusions:

LC50 ≥ 98.7 mg/l corresponding to ≥ 70 mg/l a.i.
LC0 ≥ 98.7 mg/l corresponding to ≥ 70 mg/l a.i.

Executive summary:

The acute toxicity of the test item Benzenesulphonic acid to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019).
A limit concentration of 70 mg a.i./L corresponding to a test item concentration of 98.7 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from an algae toxicity test (ErC50 (0-72 h) = 70 mg a.i./L) and a daphnia toxicity test (EC50 (0-48 h) = 100 mg a.i./L) given by the sponsor and in the current EU REACH dossier for the substance.
The study was performed over a period of 96 hours under semi -static conditions with renewal of test solutions after 48 hours of exposure. Seven test organisms were exposed to the test item 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 pH of the new test item solutions were adapted to 6.50 ± 0.1 with NaOH (1 M).The test solutions were visually clear throughout the study.
The concentration of the test item Benzenesulphonic acid was analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals (0 hours and 48 hours) and in the corresponding old media at the end of the exposure intervals (48 hours and 96 hours) in the limit test item nominal concentration and the control.
The measured concentrations of the test item were in the range of 104% to 107% of the nominal concentration in the fresh media at start of the exposure intervals and between 100% to 101% at the end of the exposure intervals.
All effect values given were based on the analytically verified nominal and the a.i. concentration of the test item.

Description of key information

LC50 for freshwater fish 96h= 70 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

70 mg/L

Additional information

The ASA Category comprises the following 5 aromatic sulphonic acids:

TSA, Toluene-4-sulphonic acid (EC 203-180-0, CAS 104-15-4)

XSA, (Xylenes and 4-ethylbenzene) sulphonic acid (EC 701-247-3, CAS -) Former EC 246-839-8

CSA, p-cumene sulphonic acid (EC 240-210-1, CAS 16066-35-6)

BSA, Benzene sulphonic acid (EC 202-638-7, CAS 98-11-3)

HBSA, 4-hydroxybenzensulphonic acid (EC No. 202-691-6, CAS No. 98-67-9)

The short-term toxicity on fish was assessed with available studies on ASA and related salt (Hydrotropes category). Hydrotropes are completely dissociated in water and could be used in read across for the related acid form.The short-term toxicity on fish has been tested on BSA TSA and HBSA. For XSA and CSA there are no studies available and the endpoint can be covered by the studies available on the corresponding salts.

A good quality (non-GLP) fish acute toxicity study is available for TSA (CAS No. 104-15-4). The 96 hr LC50 is 500 mg/L which is equivalent to 325 mg active ingredient/L based on the test substance being 65% p-toluenesulfonic acid in water.

The acute toxicity of the test HBSA to fish (zebrafish) was determined according to OECD 203. A limit concentration of 128 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (ErC50 (0-72 h) = 128 mg/L (nominal test item concentration)) and a daphnia acute immobilization test (EC50 (0-48 h) = > 152 mg/L (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. All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the measured test concentration 128 mg/L.

The acute toxicity of the test item BSA to fish (zebrafish) was determined according to OECD 203.

A limit concentration of 70 mg a.i./L corresponding to a test item concentration of 98.7 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from an algae toxicity test (ErC50 (0-72 h) = 70 mg a.i./L) and a daphnia toxicity test (EC50 (0-48 h) = 100 mg a.i./L) given by the sponsor and in the current EU REACH dossier for the substance.

The study was performed over a period of 96 hours under semi -static conditions with renewal of test solutions after 48 hours of exposure.

All effect values given were based on the analytically verified nominal and the a.i. concentration of the test item. The test item does not show toxic effects on fish at the measured test concentration 70 mg/L a.i..

For SXS LC50 96 fish is higher than 1000 mg/L, while for SCS LC50 96h fish was higher than 100 mg/L, a.i., the maximum tested concentration.

In all cases results are higher than 70 mg/l a.i., the limit concentration established by the algae toxicity test on BSA, therefore this value will be taken as conservative reference for the fish toxicity for the whole category