Bis(piperidinothiocarbonyl) hexasulphide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Deviations:

yes

Remarks:

Two concentrations tested instead of five.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A saturated stock solution of 100 mg/L was prepared in test medium. The solution was filtered. DPTH concentration was considered to be 10 µg/L as this is the value of water solubility as determined in the OECD 105 study included in the present REACh dossier. A 5 µg/L DPTH solution was prepared by dilution of the 10 µg/L solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Fertilised eggs of Danio rerio HAMILTON-BUCHANAN were used in the test.

Origin and Husbandry
The original animals were obtained from Umweltbundesamt (UBA) and used for breeding and production of eggs (In-house breeding since 2007).
Danio rerio is routinely used for breeding and toxicity tests. The fish for egg production are kept following OECD Guidelines for the Testing of Chemicals No. 236, adopted 26. July 2013 “Fish Embryo Acute Toxicity (FET) Test “.

Vessels glass aquaria
Medium chlorine-free tap water with a copper concentration be-low 0.05 mg/L (composition in annex 2 page 21)
Food three times a day; warm-water fish food and artemia
Cleaning and medium renewal at least once per week
Photoperiod 12/12 hours, using neon tubes
Temperature 26°C

For production of eggs, one day before the start of the test, the spawn dishes were placed into the aquaria and charged with two adult males and one adult female, respectively. Next morning, about 45 ± 15 minutes after lighting, the spawn dishes including the eggs were removed. After microscopical observation, the eggs which have been used in the test were separated.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25.3°C - 26.7°C min-max

pH:

7.8-7.9

Dissolved oxygen:

90.6 - 92.1% of ASV (min-max)

Salinity:

Freshwater.

Nominal and measured concentrations:

Nominal: 0 ; 5 ; 10 µg/L.
Measured: not measured.

Details on test conditions:

I - Test solution preparation
In this study, the limit of water solubility was used as highest concentrated treatment. At the beginning of the test and after 3 days a 100% saturated solution was prepared by weighing 100 mg/L (103 mg/L real load) test item (100% saturation assumed to be equal to 10 µg/L) in dilution water and using an ultrasonic bath for 85 to 90 min. This solution was filtrated through 0.45 µm PTFE filter and used to prepare a lower concentrated solution of 50% saturation (5 µg/L) by dilution with dilution water. 24.5 hours before the start of the test, the respective test solutions were prepared and the test vessels were filled with 2 mL of the respective test solution or control solutions (dilution water or positive control 3,4-DCA) and incubated in the climate chamber.

II - Description of the Study Performance
After spawning of the adult animals, the eggs were removed from the spawning aquaria and the eggs were checked with a binocular for fertilisation and the percentage of ferti-lised eggs per female were determined. Fertilised eggs can be clearly identified by the development of a blastula. The egg batch with fertilisation rate = 70% was used to fulfil validity criterion. Then, eggs were placed in dishes filled with the respective test item solution or the control solutions. The pre-conditioned test vessels were emptied and refilled with 2 mL freshly prepared test item solutions resp. controls including one viable fertilized egg per well.

II.1 - Experimental set-up
Test vessels: 24-well polystyrene plates
Duration: 96 hours
Treatments 10 / 5 µg/L nominal concentration
Observation times 24 / 48 / 72 / 96 hours post fertilization
Medium renewal after 3 days
Volume test solution: 2 mL per well
Temperature: 25.3 - 26.7°C

Treatments one plate per treatment with 20 wells filled with test solution and 4 wells filled with dilution water acting as plate (negative) control.
Controls one plate with 24 wells filled with negative control solution (dilution water)
one plate with 20 wells filled with positive control solution (4.0 mg/L Dichloroaniline) and 4 wells filled with dilution water acting as plate (negative) control

II.2 - Phys-Chem monitoring
At the start and the end of the test, pH, total hardness and conductivity were measured in the controls and in the highest test concentration. Additionally, the oxygen concentration was measured in the controls and in the highest test concentration (10 µg/L) with viable embryos at the end of the test. Measurements at the end of the test were performed by pooling of the respective well contents.


III - Biological Observations
Every 24 ± 2 hours until the end of the test all fish eggs were monitored for the following lethal parameters.

III.1 - Coagulation of Embryo
Coagulated embryos are milky white and appear dark under the microscope. The number of coagulated embryos was determined after 24, 48, 72 and 96 hours.

III.2 - Lack of Somite Formation
At 26 ± 1 °C, about 20 somites have formed after 24 hours in a normally developing zebrafish embryo. Spontaneous movements indicate the formation of somites. The ab-sence of somites was recorded after 24, 48, 72 and 96 hours.

III.3 - Non-Detachment of the Tail
In a normally developing zebrafish embryo, detachment of the tail from the yolk is ob-served following posterior elongation of the embryonic body. Absence of tail detachment was recorded after 24, 48, 72 and 96 hours.

III.4 - Lack of Heartbeat
In a normally developing zebrafish embryo at 26 ± 1 °C, the heartbeat is visible after 48 hours. Particular care should be taken when recording this endpoint since irregular heartbeat should not be recorded. Moreover, visible heartbeat without circulation in aorta abdominalis is considered non-lethal. Absence of heartbeat was recorded after 48, 72 and 96 hours.

III.5 - Other parameters
Additionally, hatching rates of all treatment and control groups were recorded from 48 hours onwards and reported. Although hatching is not an endpoint used for the calculation of the 96h-LC50, hatching ensures exposure of the embryo without a potential barrier function of the chorion, and as such may help data interpretation. Also incidence and description of morphological and physiological abnormalities, if any, were noted.

Reference substance (positive control):

yes

Remarks:

dichlorophenol

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 10 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: see remarks

Remarks:

An embryo was considered dead when at least one the following criteria was met: coalescence, lack of detachment of the tail, lack of somite, lack of heartbeat. No mortality was recorded up to the water solubility limit of the test item.

Details on results:

Embryos were considered dead if at least one of the criteria was met: coalescence, lack of somite, lack of heartbeat or non-detachment of the tail. No embryo or larvae died in the control. One embryo was found dead at 5 µg/L and none at 10 µg/L. Therefore, it can be considered that DPTH is not acutely lethal to fish up to its water solubility limit. Nevertheless, while no malformation was seen in the controls, a significant number of them was seen in the treated groups in a dose-dependent manner. However, to our knowledge, there has not been any scientific study investigating the impact of such embryo malformation on expected ulterior larval survival in Danio rerio. That is why these indicators were not considered for embryo survival assessment during the development of OECD 236 guideline. Therefore, these indicators are not predictive of what could happen on a longer term. While all control larvae hatched, hatching rate was 70% at 5 µg/L DPTH and 30% at 10 µg/L. Lack of hatching at 96h cannot be considered as embryo's death as a recent study showed that larval hatching retardation up to 6 days is not predictive of larval survival after 35 days (Sehonova et al., 2016).

Sehonova P. et al., 2016. The effect of tramadol hydrochloride on early life stages of fish. Environ. Toxicol. Pharmacol. 44, 151-157.

Results with reference substance (positive control):

Results with dichlorophenol (4 mg/L) showed 96h mortality of 45%.

Reported statistics and error estimates:

Mortality results were analysed with Toxrat (v3.2).

Sublethal observations / clinical signs:

1.1     Morphological and physiological abnormalities

Table1.1-a      Developmental abnormalities of exposed embryos after 96h

Abnormalities	Control	Positive Control	Test item (µg/L)
			5	10
Altered eye development	0	18	1	0
Present oedema	0	20	6	12
Absent pigmentation	0	9	2	10
Absent blood circulation	0	20	2	2
Head malformation	0	14	1	7
Tail malformation	0	9	4	4
Heart malformation	0	19	6	11
Spine malformation	0	2	5	4
Yolk malformation	0	1	1	0
Total affected fish	0	20	10	16

1.2     Mortality

Table1.2-a      Mortality (based on four lethal parameters) in Control

	Control																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Coagulation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No. somites	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail not detached	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No heartbeat	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table1.2-b      Mortality (based on four lethal parameters) of embryos exposed to Positive Control 3,4-Dichloroaniline at 4 mg/L

	Positive Control																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Coagulation	0	1	1	0	0	0	1	0	1	0	0	0	0	1	0	0	1	1	0	0	0	0	0	0
No. somites	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail not detached	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No heartbeat	1	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table1.2-c      Mortality (based on four lethal parameters) of embryos exposed to 5 µg/L test item

	5 µg/L																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Coagulation	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No. somites	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail not detached	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No heartbeat	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Table1.2-d      Mortality (based on four lethal parameters) of embryos exposed to 10 µg/L test item

	10 µg/L																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Coagulation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0
No. somites	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail not detached	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
No heartbeat	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

  

1.3     Morphological and physiological abnormalities

Table1.3-a      Morphological and physiological abnormalities of embryos exposed to Control

	Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Altered eye development	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Present oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Absent pigmentation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Absent blood circulation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Head malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Heart malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Spine malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Yolk malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table1.3-b      Morphological and physiological abnormalities of embryos exposed to Positive Control 3,4-Dichloroaniline at 4 mg/L

	Positive Control																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Altered eye development	1	0	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	0	0
Present oedema	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	0	0
Absent pigmentation	0	0	1	0	1	0	0	0	0	0	1	1	1	0	1	1	0	0	1	1	0	0	0	0
Absent blood circulation	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	0	0
Head malformation	0	0	1	0	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	1	0	0	0	0
Tail malformation	0	1	1	0	1	1	0	0	1	0	1	1	1	0	0	0	0	0	1	0	0	0	0	0
Heart malformation	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	0	0
Spine malformation	0	0	0	0	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0
Yolk malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0

 

Table1.3-c      Morphological and physiological abnormalities of embryos exposed to 5 µg/L test item

	50 mg/L																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Altered eye development	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Present oedema	1	0	0	0	0	0	0	0	1	0	1	1	0	0	1	0	1	0	0	0	0	0	0	0
Absent pigmentation	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0
Absent blood circulation	0	0	0	0	0	0	0	0	1	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0
Head malformation	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Tail malformation	0	0	0	0	0	0	1	0	1	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0
Heart malformation	1	0	0	0	0	0	0	0	1	0	1	1	0	0	1	0	1	0	0	0	0	0	0	0
Spine malformation	0	0	0	0	0	0	1	0	1	0	0	1	1	0	0	0	0	1	0	0	0	0	0	0
Yolk malformation	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table1.3-d      Morphological and physiological abnormalities of embryos exposed to 10 µg/L test item

	100 mg/L																				Control
Embryo	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Altered eye development	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Present oedema	1	1	1	1	1	0	1	1	1	0	1	0	0	0	1	1	0	0	0	1	0	0	0	0
Absent pigmentation	0	1	0	1	1	0	0	1	1	0	1	0	0	1	1	1	0	0	0	1	0	0	0	0
Absent blood circulation	0	0	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0
Head malformation	0	1	0	1	1	0	0	0	1	0	0	0	0	1	1	1	0	0	0	0	0	0	0	0
Tail malformation	0	0	0	1	0	0	0	0	0	0	1	0	0	0	1	1	0	0	0	0	0	0	0	0
Heart malformation	0	1	1	1	0	0	1	1	1	1	1	0	0	0	1	1	0	0	0	1	0	0	0	0
Spine malformation	0	0	0	0	0	0	0	0	0	0	1	1	0	0	1	0	0	1	0	0	0	0	0	0
Yolk malformation	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table 1.4-a       Hatching rate of exposed embryos

Nominal Conc.	Number of Embryos at the Start	Number of hatched embryos at the End (96 h)	Hatching rate in %
µg/L
Blank Control	24	24	100
Positive Control	20	9	45
5	20	15	75
10	20	6	30

 

Validity criteria fulfilled:

yes

Conclusions:

No mortality was seen up to the water solubility limit of DPTH.

Executive summary:

A fish embryo test (FET) was carried out on zebrafish according to the OECD 236 TG. A control group, a positive control group (dichlorophenol, 4 mg/L) and two DPTH concentrations were tested (5 and 10 µg/L nominal, which corresponds to the water solubility limit of DPTH according to a OECD 105 study included in this REACh dossier). The 10 µg/L solution was prepared by saturating and filtering a 100 mg/L DPTH solution. The 5 µg/L solution was prepared by dilution of the 10 µg/L solution. Zebrafish freshly spawned eggs were incubated for 96 h in test medium (20 eggs for each treatement + 4 control eggs per positive control and per treatment). Embryos were considered dead if at least one of the criteria was met: coalescence, lack of somite, lack of heartbeat or non-detachment of the tail. In addition, hatching was monitored throughout the study and a suite of malformations were evaluated (altered eye development, oedema, lack of pigmentation, lack of blood circulation, head, tail, heart, spine, yolk malformation).

No embryo or larvae died in the control. One embryo was found dead at 5 µg/L and none at 10 µg/L. Therefore, according to OECD 236 TG criteria, it can be considered that DPTH is not acutely lethal to fish embryo up to its water solubility limit.

Description of key information

A fish embryo test (FET) was carried out on zebrafish according to the OECD 236 TG. A control group, a positive control group (dichlorophenol, 4 mg/L) and two DPTH concentrations were tested (5 and 10 µg/L nominal, which corresponds to the water solubility limit of DPTH according to a OECD 105 study included in this REACh dossier). The 10 µg/L solution was prepared by saturating and filtering a 100 mg/L DPTH solution. The 5 µg/L solution was prepared by dilution of the 10 µg/L solution. Zebrafish freshly spawned eggs were incubated for 96 h in test medium (20 eggs for each treatement + 4 control eggs per positive control and per treatment). Embryos were considered dead if at least one of the criteria was met: coalescence, lack of somite, lack of heartbeat or non-detachment of the tail. No embryo or larvae died in the control. One embryo was found dead at 5 µg/L and none at 10 µg/L. Therefore, according to OECD 236 TG criteria, it can be considered that DPTH is not acutely lethal to fish embryo up to its water solubility limit.