[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-09-05 to 2016-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature, protected from light, in the tightly closed original container
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable over at least 7 days


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:none
- Final dilution of a dissolved solid, stock liquid: see "details on test solutions"

Analytical monitoring:

yes

Details on sampling:

- Concentrations: for all concentrations and the control groups.
- Sampling method: samples of test media from alternating test replicates on D-1, D0 and weekly thereafter until the end of the exposure, samples of stock solutions from freshly prepared and 7 days aged solutions of one application interval.
- Sample storage conditions before analysis: Storage of samples at room temperature until the start of the analysis. Storage of prepared samples in the autosampler at room temperature until analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: use of a solvent due to the low solubility of the test item in water: an appropriate amount of the test item was weighed in a corresponding measuring flask, filled up with solvent to the mark and treated with a magnetic stirrer for 15 minutes at approximate 1000 rpm. Other stock solutions were prepared by dilution:
- Controls:
* control : dilution water
* solvent control: 100 µL DMSO/L dilution water
- Chemical name of vehicle: DMSO
- Concentration of vehicle in test medium: loading of 100 µL/L dilution water

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: 15 - 35 adults
- Method of collection of fertilised eggs: an artificial dawning rectangular dishes (26 x 14 x 6 cm) covered with a stainless steel mesh and provided with artificial plants (plastic) was introduced into the aquaria. After approximately 1 hour the glass dishes were gently removed to collect the eggs.
- Subsequent handling of eggs: after collection, eggs were washed in dilution water and immediately transferred into vessels containing the respective exposure solutions without regard to fertilization (eggs fully covered with the respective test solutions) = start of exposure.
After approximately 2 h post fertilization, eggs were checked for fertilization. Under a stereo microscope every embryo was checked for its blastomer phase. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and are regarded to be fertilized. Eggs with only a 2 cell blastomer are regarded as not to be fertilized. These eggs as well as coagulated eggs were discarded.

POST-HATCH FEEDING
- Start date: starting 2 days after begin of hatch (equivalent to study D5 or post-hatch D0)
- Type/source of feed:
* Larvae first fed with starter food (e.g. TetraMin Baby Hauptfutter (TETRA GMBH, D-49304 Melle, Germany))
* One day after start of feeding larvae were additionally fed with brine shrimp nauplii (48 h old) until the end of the test (1 – 9 times daily).
- Frequency of feeding:1-9 times daily.

Test type:

flow-through

Water media type:

other: tap-water filtered on activated charcoal to remove residual chlorine

Remarks:

water analysed on an annual basis by the local supplier

Limit test:

no

Total exposure duration:

30 d

Remarks on exposure duration:

post-hatch

Hardness:

Controls: mean = 64 mg CaCO3/L (66 - 68 mg CaCO3/L)
At 40 µg/L: mean = 68 mg CaCO3/L (67-68 mg CaCO3/L)
At 250 µg/L: mean = 67 mg CaCO3/L (66-68 mg CaCO3/L)

Remark: because of the mortality rate in the test concentrations 100 and 250 µg/L on study D8 and D9, the measurement was carried out from the next lower concentration 40.0 µg/L from there.
Erroneously, no measurement was carried out on study D21 from the test concentration 40.0 µg/L, however the other water parameters were analysed.

Test temperature:

Recorded continuously (once per hour) with a data logger: mean +/- SD T°C through the study = 25.8 +/- 0.4 °C with a minimum of 24.6 °C and a maximum of 26.7°C.

pH:

Controls: mean+/- SD = 7.84 +/-0.15 (7.63 - 7.95)
Solvent controls: mean +/- SD = 7.90 +/- 0.107 (7.75 - 8.03)
At 6.40 µg/L: mean +/- SD = 7.91 +/- 0.09 (7.77 - 8.01)
At 16.0 µg/L: mean +/- SD = 7.89 +/- 0.095 (7.74-7.99)
At 40.0 µg/L: mean +/- SD = 7.89 +/- 0.069 (7.78-7.97)
At 100 µg/L: mean +/- SD = 8.00 +/- 0.014 (7.99-8.01)
At 250 µg/L: mean +/- SD = 8.00 +/- 0.007 (7.99-8.00)

Dissolved oxygen:

Controls: mean+/- SD = 96 +/- 2.59 (92-100)
Solvent controls: mean +/- SD = 96 +/-2.37 (92-99)
At 6.40 µg/L: mean +/- SD = 96 +/-2.68 (92-100)
At 16.0 µg/L: mean +/- SD = 96 +/- 2.65 (92-100)
At 40.0 µg/L: mean +/- SD = 96 +/- 2.02 (93-100)
At 100 µg/L: mean +/- SD = 98 +/-1.48 (96-100)
At 250 µg/L: mean +/- SD = 97 +/- 3.65 (91-100)

Conductivity:

Conductivity of the tap water 175 µS/cm (measured quarterly)

Nominal and measured concentrations:

Nominal concentrations: 6.40 - 16.0 - 40.0 - 100 - 250 µg/L
Measured concentrations: the measured concentrations of the test media were in the range of 83% to 112% of the nominal values

Details on test conditions:

TEST SYSTEM
- Test vessel:
* Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm).
* Test vessels were covered by glass lids.
* Volume of the test media of approximately 7.5 L.
* Establishment of a randomized block design with each treatment being present in each block
- Aeration: dilution water supply tank aerated. No additional aeration of the test vessels provided
- Type of flow-through: peristaltic
- Equilbration period: test solutions flowed through test vessels for 8 days prior to the initiation of the exposure.
- Renewal rate of test solution (frequency/flow rate): mean flow rate though the mixing chambers (test item and controls) = 8.94 +/- 0.322 L/h (8.16 - 9.60)
- No. of fertilized eggs/embryos per vessel: 20 eggs
- No. of vessels per concentration: 4 replicates
- No. of vessels per control: 4 replicates
- No. of vessels per vehicle control: 4 replicates
- Biomass loading rate:
* Based on the 7.5 liter volume of a single growth chamber, the biomass-loading was 104 mg/L.
* Based upon a flow of 52.5 liters per day through each single test chamber, the biomass-loading was 2 mg per liter and day.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water filtered on activate charcoal to remove residual chlorine.
- Total organic carbon: mean TOC of the dilution water throughout exposure, determined on study days 0, 8, 15, 21, 28 and 34 was 1.48 mg/L
- Chlorine: residual chlorine, measured of the dilution water on study days 0, 7, 14, 19, 27 and 34 was < 0.01 mg/L.
- Alkalinity: Alkalinity of the tap water 0.80 mmol/L (measured quarterly)
- Intervals of water quality measurement:
* Temperature: continously
* Dissolved oxygen and T° in one replicate of each test group: at least 3 times per week
* Flow rates of the test media: at least 3 times per week
* pH value in alternate replicates: weekly
* TOC from dilution water: weekly
* Chlorine from dilution water: weekly
* Total hardness in one replicate of the control and the highest test item concentration: weekly
- Cleaning: test medium siphoned as needed to remove excess faecal material and uneaten food. The mesh coated fittings were clean daily after starts of feeding.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: mean = 370 lux (from 190 to 575), measured at the start of exposure on the surface of the test vessels.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hatching: number of hatched larvae was determined daily until study day 7
- Eggs/embryos mortality: checked daily: if fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded
- Larvae and juvenile fish mortality: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.

- Abnormal appearance and behavior (abnormality of the body, hyperventilation, uncooridnated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior). Recorded by visually inspecting each replicate at adequate interval.
- Measurement of fish size: at the end of exposure (post-hatch D30)
- Measurement of fish wet weight: at the end of exposure (post-hatch D30)

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 10, 100 and 1000 µg/L (nominal). Measured concentrations were in the range of 94 to 102 % of the nominal values. Performed on 2 replicates of 20 eggs under flow through conditions.
- Results used to determine the conditions for the definitive study:
* 1000 µg/L: Hatch from D3 to D6 = 83% and mortality after 9 days = 100 %
* 100 µg/L: Hatch from D3 to D6 = 83% and mortality after 9 days = 38 %
* 10 µg/L: Hatch from D3 to D6 = 93% and mortality after 9 days = 8 %
* Control: Hatch from D9 = 95% and mortality after 9 days = 5 %

POST-HATCH DETAILS
- Begin of post-hatch period: Study D5 (PDH0)

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 700 eggs
- Removal of eggs to check the embryonic development on day no. 1
- Fertilization rate: 95%

Reference substance (positive control):

not required

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

ca. 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post hatch survival and overall survival

Remarks on result:

other:

Key result

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

ca. 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post hatch survival and overall survival

Duration:

35 d

Dose descriptor:

LC50

Effect conc.:

ca. 49.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post hatch survival and overall survival

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 250 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 250 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 250 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

> 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

> 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Fry (post-hatch) Mortality:
* Control: mean 16 % mortality
* solvent control: mean 16 % mortality
* Test group 6.40 µg/L: 2% mortality (no statistically significant difference from control groups)
* Test group 16.0 µg/L: 13% mortality (no statistically significant difference from control groups)
* Test group 40.0 µg/L: 15% mortality (no statistically significant difference from control groups)
* Test group 100.0 µg/L: 100% mortality (statistically significant difference from control groups)
* Test group 250.0 µg/L: 100 % mortality (statistically significant difference from control groups)

- Overall mortality (at the end of the study)
* Control: mean 16 % mortality
* solvent control: mean 17 % mortality
* Test group 6.40 µg/L: 4% mortality (no statistically significant difference from control groups)
* Test group 16.0 µg/L: 15% mortality (no statistically significant difference from control groups)
* Test group 40.0 µg/L: 16% mortality (no statistically significant difference from control groups)
* Test group 100.0 µg/L: 100% mortality (statistically significant difference from control groups)
* Test group 250.0 µg/L: 100 % mortality (statistically significant difference from control groups)

- Number of fish in swim-up stage at one or more time periods: observation for a 5-day period from study D4 to D8.
Mean value for the 4 replicates for each study days
* Control group: D4 = 0%; D5 = 95%; D6 = 99%; D7= 99%; D8 = 100%
* Solvent control group: D4 = 0%; D5 = 95%; D6 = 99%; D7 and D8 = 100%
* Test group 6.40 µg/L: D4 = 1%; D5 = 95%; D6 to D8 = 100%
* Test group 16.0 µg/L: D4 = 3%; D5 = 95%; D6 to D8 = 100 %
* Test group 40.0 µg/L: D4 = 0%; D5 = 98%; D6 to D8 = 100 %
* Test group 100.0 µg/L: D4 = 0%; D5 = 45%; D6 = 79% [D7 and D8 = not applicable since lethal and non-lethal effects interfered with the swim up behavior]
* Test group 250.0 µg/L: D4 = 0%; D5 = 8%; D6 = 20% [D7 and D8 = not applicable since lethal and non-lethal effects interfered with the swim up behavior]


- Observations on body length and weight of young at one or more time periods:
At study D35 (post-hatch D30, mean +/- SD total LENGTH per fish:
* Control group: 16.7 mm +/- 0.750
* Solvent control group: 16.8 mm +/- 0.450
* Test group 6.40 µg/L: 15.9 mm +/- 0.206 (no statistically significant difference from control groups)
* Test group 16.0 µg/L: 16.9 mm +/- 0.658 (no statistically significant difference from control groups)
* Test group 40.0 µg/L: 16.3 mm +/- 0.739 (no statistically significant difference from control groups)
* Test group 100 µg/L: no survival at the end of the test
* Test group 250 µg/L: no survival at the end of the test

At study D35, mean +/- SD total WEIGHT per fish:
* Control group: 0.0390 mg +/- 0.003
* Solvent control group: 0.0390 mg +/- 0.004
* Test group 6.40 µg/L: 0.037 mg +/- 0.0002 (statistically significant difference from control groups, however, as per definition of the LOEC all test concentrations above the LOEC must have a harmful effect or greater, this statistical significance of the lowest test concentration is considered to be negligible)
* Test group 16.0 µg/L: 0.0384 mg +/- 0.004 (no statistically significant difference from control groups)
* Test group 40.0 µg/L: 0.0375 mg +/- 0.003 (no statistically significant difference from control groups)
* Test group 100 µg/L: no survival at the end of the test
* Test group 250 µg/L: no survival at the end of the test

- Number of healthy fish at end of test: all survival fish were healthy at the end of the test.

- Type of and number with morphological abnormalities: none
- Type of and number with behavioural abnormalities:none
- Type and number of developmental effects: no develpmental effects
- Other biological observations: no other biological observations
- Effect concentrations exceeding solubility of substance in test medium: the substance is insoluble in water, therefore DMSO was used.
- Incidents in the course of the test which might have influenced the results:- Mortality/survival at embryo, larval, juvenile, and adult stages: 100% of mortality in the test groups 100 µg/L and 250 µg/L, therefore, not all paramaters where determined for this 2 concentrations test.
- Days to hatch: Hatch began on study D3 in the controls and all test item concentration and continued until study D7 (at study D5, the hatching rate was of 95% in the control and 98% in the solvent control).

Results with reference substance (positive control):

not required

Reported statistics and error estimates:

Statistical analyses performed with TOXRAT SOLUTIONS GMBH software.

*Control group comparison : Fisher`s Exact Binomial Test (significance level 0.05, for the parameter hatchability, post hatch survival and overall survival) and Student-t Test (significance level 0.05, for the parameter fry growth (expressed as length and fresh weight)).

Following negative results both controls were pooled for analysis.

*For parameters analysed: hatch on study days 4, 5, 6 and 7, post hatch survival, overall survival and fry growth (expressed as length and fresh weight):

Monotonicity : done by trend analysis by contrasts (with P<0.05).
Chi2 2x2 table test with Bonferroni correction (with P<0.05 and P<0.01) -->
Tarone’s Test : (with P<0.01)
Step-down Cochran Armitage Test : (with P<0.05).
Shapiro-Wilk’s : (with P<0.01).
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm : (with P<0.05).
Dunnett’s Multiple t-test procedure : (with P<0.05).

Validity criteria fulfilled:

yes

Conclusions:

The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations ≥ 100 µg/L.
For the parameter hatchability, the NOEC (nominal, PHD 0 – 30) was ≥ 250 µg/L. Therefore the LOEC for hatchability (nominal, PHD 0 – 30) was determined to be > 250 µg/L.
For the parameters post-hatch and overall survival (mortality), the NOEC (nominal, PHD 0 – 30) was 40.0 µg/L. Therefore the LOEC for these parameters (nominal, PHD 0 – 30) was determined to be 100 µg/L.
For the parameter fry growth (expressed as length and fresh weight) the NOEC (nominal, PHD 0 – 30) was ≥ 40.0 µg/L. Therefore the LOEC for these parameters (nominal, PHD 0 – 30) was determined to be > 40.0 µg/L.

All effect values are given based on the nominal concentrations of test item, since the measured concentrations of the test media were in the range of 83% to 112%

Executive summary:

The effects of the test item to the early-life stage of fish (Danio rerio/ Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2016-10-27 to 2016-12-01.

 

A test under flow-through conditions was conducted with the nominal test item concentrations of CHESAR 6.40 - 16.0 - 40.0 - 100 - 250 µg/L. Due to the low solubility of the test item in water, DMSO was used as solvent with a loading of 0.100 mL/L dilution water.

 

The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio/zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).

 

The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.

On study day five, 95% of the control and 98% of the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).

 

Different toxicological endpoints were determined: hatch, time to hatch, fry growth (expressed as length and fresh weight), morphological and behavioral effects, post-hatch survival and overall survival (mortality).

 

Specific analysis of various concentrations of test item in the test media and the control was carried out via LC-MS/MS. The analytical method has been validated according to SANCO 3029/99 rev.4 (2000).

 

The test media were sampled and analysed prior to exposure on day -1 and during the exposure on study days 0, 7, 15, 22 and 28. The measured concentrations of the test media on study days 0 to 28 were in the range of 83% to 112% of the nominal values.

The stock solutions were sampled and analysed on study day 15 and 22 from freshly prepared and corresponding 7 days aged stock solutions, respectively. Measured concentrations of the stock solutions were in the range of 89 to 100% of the nominal values, demonstrating stability over at least seven days.

All effect values are given based on nominal concentrations of the test item (see Table 1 and Table 2).

Findings and Observations

The results of the parameters hatch, time to hatch, fry growth (expressed as weight and length), post hatch survival and overall survival were checked for statistically significant differences.

The effect values NOEC and LOEC were determined based on the statistical results. The results are presented in the tables below:

Table 1: NOEC, LOEC, ECx Values of Hatchability and Fry Growth (based on nominal test item concentrations [µg/L])

Parameter	Hatchability	Fry Growth express as
		Length	Weight
NOEC [µg/L]	≥250	≥40.0	≥40.0
LOEC [µg/L]	> 250	> 40.0	> 40.0
EC10 (95 % C.I.)	> 250 (not determinable)	> 40.0 (not determinable)	> 40.0 (not determinable)
EC20 (95 % C.I.)	> 250 (not determinable)	> 40.0 (not determinable)	> 40.0 (not determinable)
EC50 (95 % C.I.)	> 250 (not determinable)	> 40.0 (not determinable)	> 40.0 (not determinable)

C.I.= Confidence interval

Table2:   NOEC, LOEC, LC_xvalues of Post Hatch Survival and Overall Survival (based on nominal test item concentrations [µg/L])

 

Parameter	Post hatch survival	Overall survival (mortality)
NOEC [µg/L]	40.0	40.0
LOEC [µg/L]	100	100
EC10 (95 % C.I.)	35.7 (< 6.40 – 76.1)	32.1 (< 6.40 – 77.7)
EC20 (95 % C.I.)	42.0 (28.6 – 77.1)	41.8 (27.6 – 78.7)
EC50 (95 % C.I.)	49.1 (40.9 – 81.0)	49.1 (40.8 – 82.6)

Conclusions

The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations ≥ 100 µg/L.

For the parameter hatchability the NOEC (nominal, PHD 0 – 30) was ≥ 250 µg/L. Therefore the LOEC for hatchability (nominal, PHD 0 – 30) was determined to be > 250 µg/L.

For the parameters post hatch and overall survival (mortality) the NOEC (nominal, PHD 0 – 30) was 40.0 µg/L. Therefore the LOEC for these parameters (nominal, PHD 0 – 30) was determined to be 100 µg/L.

For the parameter fry growth (expressed as length and fresh weight) the NOEC (nominal, PHD 0 – 30) was ≥ 40.0 µg/L. Therefore the LOEC for these parameters (nominal, PHD 0 – 30) was determined to be > 40.0 µg/L.

Description of key information

NOEC for freshwater fish (Danio rerio)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

40 µg/L

Additional information

For the parameters post hatch and overall survival (mortality) the NOEC (nominal, PHD 0 – 30) was 40.0 µg/L.

For the parameter hatchability the NOEC (nominal, PHD 0 – 30) was ≥ 250 µg/L.

For the parameter fry growth (expressed as length and weight) the NOEC (nominal, PHD 0 – 30) was ≥ 40.0 µg/L.