Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.02. - 08.11.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

EC Number:

225-935-3

EC Name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Cas Number:

5160-02-1

Molecular formula:

C17H13ClN2O4S.1/2Ba

IUPAC Name:

barium(2+) bis(5-chloro-2-[(1E)-2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-4-methylbenzene-1-sulfonate)

Test material form:

solid: nanoform

Specific details on test material used for the study:

Name of test substance: Pigment Red 53:1
CAS No.: 5160-02-1
Homogeneity: Given
Physical state/Appearance: Solid / red
Density: 1.73 g/cm3

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling of the complete dispersion:
Analytical concentration controls were conducted for the calibration phase (non-GLP analysis), start of exposure prior the insertion of daphnids (study day 0) and each stock-dispersion renewal thereafter. For more information on sample storage conditions see sampling schemes below.

Test solutions

Vehicle:

no

Details on test solutions:

Separate stock dispersions were prepared for each test concentration. For each concentration a 3L-stock dispersion was prepared.
The dispersions were prepared from sonicated test substance concentrates and made up with purified water. Each concentrate was prepared by weighing the respective amount of test substance into a rosette-cell (RZ 3, Bandelin, Germany) and adding 60 mL of purified water. Each concentrate was sonicated in an ultrasonic bath for approximately 45-60 minutes to ensure a thorough wetting of the test substance. Afterwards, each concentrate was sonicated by applying an energy of approx. 3 kJ/mL (corresponds in total to approx. 180kJ) with a BANDELIN Sonopuls HD4400 unit equipped with a TS413-Sonotrode, set to an amplitude of 30%. Finally, each concentrate was transferred into a volumetric flask and a respective amount of purified water was added. The stock dispersions were stirred continuously.
Acceptable test solution pH was within the range of 6 - 9. If a test solution was outside of this range, it was adjusted to control media pH (±0.2) with HCl or NaOH prior to the start of exposure. No adjustment of test solution pH was done prior to or during the exposure period.
The stock used for the preparation of the control test solutions was prepared under the conditions of the test-substance dispersions, i.e., 60 mL of purified and sonicated water was transferred into a volumetric flask and filled up to 3L with purified water. The test medium was stirred until its use.
Fresh stock dispersions were prepared once weekly.
All test solutions containing test substance were visibly colored reddish throughout the exposure period. No undissolved test particulate material was visible.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.
- Feeding during test: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1010 mg TOC/L and 1180 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤495 μL) of the concentrate/test vessel/day to reach a constant rate of 0.1 mg TOC/Daphnia/day. The algae were stored in a refrigerator
(dark, about 4-8°C) for maximum 21 days.
By adding the algal concentrate, the test solution was slightly diluted. During one day with a total addition of max. 0.495 mL algae concentrate added to 250 mL test solution, would result in a maximal dilution of 0.2%.

ACCLIMATION
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

2.20 - 3.20 mmol/L

Test temperature:

18.2-19.7°C

pH:

7.5 - 8.5

Dissolved oxygen:

≥ 3mg/L

Conductivity:

550–650 μS/cm

Nominal and measured concentrations:

0 (control), 0.63, 1.3, 2.5, 5.0, 7.5 mg/L as nominal concentrations based on test-substance mass without correction for content or purity.


0 (control), 0.68, 1.33, 2.33, 4.57, and 7.81 mg/L as mean analytically measured concentrations (time-weighted mean).

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Flow-Through-Chamber (height: 105 mm, inner diameter: 60 mm) equipped with a glass inlet (height: 90 mm, inner diameter: 58 mm) secured with a wire gauze (mesh size: 100 μm) at one end to prevent the passing of the daphnids.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): Flow-through, approx. 3-fold test solution renewal per day, in order to ensure constant exposure over the test period.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.02 animals / mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes.
- Intervals of water quality measurement: The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH, hardness, and temperature were measured once per week in one altering replicate test chamber for the control and the test groups. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. The pump rate of each 1-channel peristaltic pump was determined once weekly.
Light intensity was measured once during the study.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h darkness
- Light intensity: About 623-685 lux at a wave length of 400-700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every working day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each parent for the duration of the study. Separating neonates from adults was accomplished by gently removing juvenile daphnids from each chamber using a pipette and counted on a light table before being discarded. Since the daphnia will be held in groups, reproductive output will be expressed as ‘total number of living offspring produced per surviving parent.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily.

Reference substance (positive control):

yes

Remarks:

sodium chloride (NaCl)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

4.57 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The exposure of the test organism to the test substance resulted in a non-monotonic dose-response relationship. Hence, expressing the toxic effect of the test substance on the organisms' reproductive output as ECx by fitting the data to an appropriate model by non-linear regression would not provide useful data. Therefore, the toxic effect of the test substance is solely expressed as the NOEC/LOEC value. The guideline 211 acknowledges the use of the NOEC/LOEC approach as ECx alternative. The calculation of the EC10 was done nevertheless and can be found in the Appendix of the study report.
Due to the exposure concentration deviations >20% from the nominal test concentration in the 7.5 mg/L treatment, the interpretation of the test results should be based on and expressed as time-weighted mean concentrations, according to OECD 211 guideline recommendations.

No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.
The toxicity results presented here are consistent with the results from preliminary tests.
The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Over the course of the study particle size measurements for the stock dispersions containing test substance revealed a mean z-average between 204–237 nm with a corresponding mean polydispersity index between 0.15–0.26, respectively. In the control, the comparatively high mean z-average of 2598 nm (mean polydispersity index: 1.31) can most likely be considered a result of the absence of particles producing unreasonable and non-reliable results, leading to a weak scattering of the particles and noisy results, disabling a valid measurement.
Once the stock dispersions were mixed with the test medium the mean z-average visibly increased, most likely due to the higher ionic strength of the test medium compared to the purified water the stock dispersions were prepared in, inducing an agglomeration of the present particles. The control and the lowest tested concentration (0.63 mg/L) showed comparatively high particle sizes, relative to the test groups with higher test substance concentrations, most likely due to insufficient particle concentrations, leading to a weak scattering of the particles aggravating the accuracy of the measurement, that ultimately disabled a valid measurement.
When the mixing of stock dispersion and test medium was finished and after transfer to the test vessels, the particle size was increased once more, to a mean z-average of approx. 500 nm (except for the control and test group 1), probably as a result of ongoing agglomeration of the particles when applied to the test medium. However, the mean z-average in test group 2–5 reached a plateau at approx. 380–580 nm over the course of the study. Likewise, for the test vessels the control and test group 1 showed comparatively high particle sizes, relative to the test groups with higher test substance concentrations, most likely due to insufficient particle concentrations, leading to a weak scattering of the particles aggravating the accuracy of the measurement, that ultimately disabled a valid measurement.
Therefore, the test substance can be considered partly stable when dispersed in the test medium under testing conditions. The increasing mean z-average demonstrates agglomeration of the particulate test substance fractions over time. The dispersion stability of the test substance in the OECD 211 and 221 test media was intermediate (see Table 3), which is in line with the dispersion stability results of OECD 318 (see Chapter 5.6).

Any other information on results incl. tables

Tab. 1: Mortality and Reproduction Summary after 21 days.

 

Test groups	Nominal concentration [mg/L]	Mortality   Parent animals	Reproduction per surviving parent
			Mean Living Young	% effectb
0	0 (control)	0	64.3 (6.07%a)	–
1	0.63	0	54.5	–
2	1.3	0	49.4	–
3	2.5	0	59.0	–
4	5	0	45.5	29.24**
5	7.5	0	48.1	25.17**
–: Not measured or evaluated. a: Coefficient of variation for control fecundity based on surviving parents. b: Effect relative to control. Only calculated for statistically significant effects.

Statistical significance: *= p≤ 0.05, **= p≤ 0.01.

 

 

Tab. 2: Other biological observations among surviving parent animals after 21 days:

 

Nominal concentration [mg/L]	Mean growth (length, mm)	% Immobile young	Mean days to first brood	% Aborted eggs
0 (control)	3.63	0	9	0
0.63	3.67	0	9.5	0
1.3	3.66	0	9.3	0
2.5	3.66	0	9	0
5	3.58	0	9	0
7.5	3.66	0	9	0

Statistical significance: *= p≤ 0.05, **= p≤ 0.01.

 

On study day 5-6 a daphnid appeared to be small in replicate 3 of test group 1 (Control) in comparison to the remaining animals of the Control. This appearance was transient as it was not observed anymore over the remaining exposure period. No additional adverse effects or abnormal behavior were observed in any of the other test groups.

 

Table 3: Full results of the organic pigment dispersion stability in the OECD 201, 211 and 221 media.

Pigment Name	Test Medium	Stability after 6h, %	Standard deviation, %	Stability after 15h, %	Standard deviation, %	Stability after 24h, %	Standard deviation, %
Pigment Red 53:1	OECD 210	98.4	0.1	95.8	0.2	93.2	0.7
	OECD 211	75.7	0.9	44.7	2.5	24.3	2.3
	OECD 221	95	0.4	76.7	4.2	59.5	3.5

 

Table 4: Dissolution behavior of the test substance in the test water.

Day	0 mg/L (control)	0.63 mg/L	1.3 mg/L	2.5 mg/L	5 mg/L	7.5 mg/L
0	0	1	1	1	1	1
1	0	1	1	1	1	1
2	0	1	1	1	1	1
3	0	1	1	1	1	1
4	0	1	1	1	1	1
5	0	1	1	1	1	1
6	0	1	1	1	1	1
7	0	1	1	1	1	1
8	0	1	1	1	1	1
9	0	1	1	1	1	1
10	0	1	1	1	1	1
11	0	1	1	1	1	1
12	0	1	1	1	1	1
13	0	1	1	1	1	1
14	0	1	1	1	1	1
15	0	1	1	1	1	1
16	0	1	1	1	1	1
17	0	1	1	1	1	1
18	0	1	1	1	1	1
19	0	1	1	1	1	1
20	0	1	1	1	1	1
21	0	1	1	1	1	1

Explanation of abbreviations:

0: no remarkable observations, clear test medium

1: reddish colored dispersion

 

Table 5:Analytical Concentration Control

Nominal Concentration [mg/L]	Renewal period	Analytically Measured Concentration
		Initial [mg/L]	24h old [mg/L]	Time-weighted Mean
				[mg/L]	[%]a
0 (Control)	Day 0 - 4	<LOQ	<LOQ	-	-
	Day 4 - 11	<LOQ	<LOQ
	Day 11 - 18	<LOQ	<LOQ
0.63	Day 0 - 4	0.71	0.71	0.68	108%
	Day 4 - 11	0.71	0.62
	Day 11 - 18	0.62	0.74
1.30	Day 0 - 4	1.28	1.55	1.33	102%
	Day 4 - 11	1.55	1.29
	Day 11 - 18	1.29	1.11
2.5	Day 0 - 4	2.35	2.01	2.33	93%
	Day 4 - 11	2.01	2.51
	Day 11 - 18	2.51	2.49
5.0	Day 0 - 4	4.50	4.56	4.57	91%
	Day 4 - 11	4.56	4.49
	Day 11 - 18	4.49	4.76
7.5	Day 0 - 4	7.42	7.69	7.81	104%
	Day 3 - 4	7.56b	25.19
	Day 4c	25.19	15.92
	Day 4c	15.92	8.20
	Day 4c	8.20	7.69
	Day 4 - 11	7.69	7.55
	Day 11 - 18	7.55	7.16

a: % of Nominal concentration

b: Average of Day 0-4 Initial and 24 old

c: Additional samples, taken, and analyzed after momentary failure of the dosing unit, that lead to a temporarily increased test substance concentration in the respective test group

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic invertebrates.