2[2'-Hydroxy-5'-(phenyl)ureylenphenyl]benzothiazole

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:

27 May 2019 - 15 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 23: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals

Version / remarks:

Feb 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Remarks:

UPLC-FLR

Details on sampling:

Samples for analysis were taken from all test concentrations and the controls and stock solutions.
Sampling:
Frequency One day before the start of exposure to check the functioning of the system. At the start, after 7, 14 and 21 days of exposure.
Volume: 2.0 mL
Storage: If not analysed on the day of sampling samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 2.0 mL were taken from test solutions, controls and stocks for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for this a maximum of three months after delivery of the draft report, pending the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

The batch of test subsatnce tested was a slightly yellow powder with a purity of 98%.
No correction was made for the purity/composition of the test substance.
Stock solutions were prepared in Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) at a concentration of 100 mg/L.
No other treatment than vigorous shaking was needed in order to completely dissolve the test item in solvent.
Due to the possible photosensitivity of the test item, preparation of test solutions during the reproduction test was performed under dimmed light conditions and amber glassware was used in the reproduction test.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus,1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection This system has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: To initiate the test, young daphnids < 24 hours bold were selected, from parental daphnids greater than two weeks old.

Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae
Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Remarks on exposure duration:

As per the test guideline.

Post exposure observation period:

No post exposure observation period specified in the study report.

Test conditions

Hardness:

180 mg/L expressed as CaCO3 and the pH between 6 and 9. Total hardness complied with the requirements as laid down in the study plan (>140 mg CaCO3 per liter).
Hardness (mg CaCO3) range: 179-196

Test temperature:

The temperatures generally complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C). The temperature measured in control vessel slightly exceeded the range (max. 23°C)
Temperature measured continuously in control vessel (°C) range: 20-23

pH:

The pH was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).
pH range: 7.9-8.1

Dissolved oxygen:

The oxygen concentration complied with the requirements as laid down in the study plan (> 3 mg/L).
Dissolved oxygen concentration (mg O2/L) range: 8.3-9.2

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

Measured Concentrations: The concentration measured at the start of the test was 10 μg/L. Concentrations measured at weekly intervals decreased with the time of exposure (i.e. were 8.1, 6.5 and 6.6 μg/L at day 7, 14 and 21 of the exposure, respectively). This decrease can be associated with the growth of daphnids, presence of offspring in test medium and accumulation of the algal biomass.
The average exposure concentration was calculated to be 7.8 μg/L which is 78% of the target concentration. All measured concentrations were above the water solubility limit of the test item as determined in Test Facility Study No. 20218324. This was because of the DMSO used in the stock treatment solutions, which would increase the solubility of the test substance relative to pure water solution.

Details on test conditions:

Test item: 10 μg/L1
Controls: Test medium without test item or other additives (blank control) and one control containing test medium with the additives used in the treatment of the stock solutions (solvent control).

Test Procedure and Conditions
Test duration: 21 days
Test type: Flow-through, with continuous renewal of test media
Test vessels: Stainless steel with a medium volume of 1.7 L containing four mesh containers. Covered with a Perspex plate.
Medium: M7

Experimental design:
At the start of the experiment (nominal day 0) twenty neonate daphnids, less than one day old, per group were divided over four stainless steel mesh containers (five daphnids per mesh).
Light: 16 h photoperiod daily, with intensity ~400 lux.
Feeding: Daily, a quantity of a Chlorella sp. suspension3 is added as feed for the daphnids providing a ration of ca. 0.4 mg C/daphnid/day.

Sampling for Analysis of Test Concentrations

Samples for analysis were taken from all test concentrations and the controls and stock solutions. The method of analysis is described in the appended Analytical Report

Sampling:
Frequency: One day before the start of exposure to check the functioning of the system. At the start, after 7, 14 and 21 days of exposure.
Volume: 2.0 mL
Storage: If not analysed on the day of sampling samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 2.0 mL were taken from test solutions, controls and stocks for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending the decision of the sponsor for additional analysis.

Measurements and Recordings

Parental daphnids
Condition: Every day, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every day.
Body length: At the end of the test.

Offspring
Appearance first brood: When observed.
Newborn daphnids: Every day, the number of new-born young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs When observed.
Incidence of immobility: When observed.

Test medium
Temperature and pH: Once a week in all test solutions. Additionally, the temperature was continuously monitored in a temperature-control vessel.
Dissolved oxygen: Twice a week
Hardness: Once a week in both control groups and the highest test concentration.
Light: At the start and the end of the test.

INTERPRETATION
Acceptability of the Test
The mortality of the parent animals (female Daphnia) in the control did not exceed 20% at the end of the test.
The average cumulative number of young per female in the controls after 21 days was ≥ 60.
Data Handling
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
The values for reproduction observed at various concentrations of the test item were expressed as mean number of living young per introduced parent4. The cumulative values for reproduction at each concentration were compared to those recorded in the control (day 21 of exposure). Further, the lengths of the parental daphnids (day 21) exposed to the test item were compared with the control.

ANALYSIS
Exposure concentrations: The exposure concentrations were calculated as an arithmetic mean of all values.
Statistical analysis: Differences between control treatments were analysed with Student t-test for homogenous variances.
As there was a significant difference in reproduction between both control treatments, the comparison of reproduction in the limit concentration was done with the solvent control. For consistency, all other comparisons were performed with solvent control.
The following statistical procedures were used to determine the NOEC for reproduction and
growth:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Differences between treatments and the control: Two-sample t-test procedure.
Mortality of parental daphnids:
• Both the differences between control treatments and the difference between limit concentration and control were analysed with Fisher`s Exact Binomial Test.
All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH,Germany).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.8 µg/L

Basis for effect:

reproduction

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 7.8 µg/L

Basis for effect:

reproduction

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.8 µg/L

Basis for effect:

growth

Details on results:

Stability test
The concentration measured in freshly prepared solution was higher than the target concentration, i.e. was 14 instead of 10 μg/L. The measured concentrations in both vessels (with daphnia and algae (biotic) and without organisms (abiotic)) decreased significantly in time. The decrease in abiotic vessel was stronger than in the biotic vessel. The exact reason is unknown but possibly, some portion of light was absorbed by algae in the biotic vessel providing shielding for the test item. As the test item is possibly sensitive to light it was decided to keep light intensity as low as possible during the reproduction test.
As a decrease of 56-67% was observed after the first 24 hour of incubation, it was decided to perform the reproduction test under flow-through instead of semi-static conditions.

Reproduction Test
Measured Concentrations
The concentration measured at the start of the test was 10 μg/L. Concentrations measured at weekly intervals decreased with the time of exposure (i.e. were 8.1, 6.5 and 6.6 μg/L at day 7, 14 and 21 of the exposure, respectively). This decrease can be associated with the growth of daphnids, presence of offspring in test medium and accumulation of the algal biomass.
The average exposure concentration was calculated to be 7.8 μg/L which is 78% of the target concentration. All measured concentrations were above the water solubility limit of the test item as determined in Test Facility Study No. 20218324. This was because of the DMSO used in the stock treatment solutions, which would increase the solubility of test item relative to pure water solution.

Time to First Reproduction
First offspring were observed in all groups at day 7, therefore, the test item did not affect the onset of reproduction

Reproduction
On average 175 offspring were produced per parental daphnid in the blank and 136 in then solvent control. Hence, the validity criterion of at least 60 offspring per parental daphnid was
met. As the difference in reproduction between the blank and solvent control was statistically significant, the latter was used for statistical analysis of effects in the test item treated group.
A stimulation of reproduction was observed in the test item treated group when compared with solvent treatment (14% increase5). As only a reduction of reproduction is considered a relevant effect, the NOEC for reproduction was 7.8 μg/L
Six immobile offspring were observed in the blank control treatment, while none were recorded in the solvent control or in the test item treated group. Five aborted eggs were observed in the test item treated group.

Body Length
No statistically significant differences were found between controls. For consistency, the effect in the limit group was compared to the solvent control; no statistically significant effects were found . Therefore, the NOEC for growth was 7.8 μg/L.
In each of the control treatments 2 of the 20 exposed daphnids did not survive exposure, hence the mortality of parental daphnids did not exceed 20% and complied with the validity criteria of the study.
At day 1 of exposure, 5 daphnids were observed to be trapped at the surface of test solution in the limit concentration (3 in replicate B and 2 in replicate D). These daphnids were subsequently submerged in test solution. On the following day, one daphnid was still observed to float at the surface (replicate B) and was scored as immobile. Next day (day 3) one daphnid in this replicate was observed to be dead. It was considered the same daphnid as the immobilised one on day 2 and therefore, its mortality was counted as occurred on day 2.
The mortality observed at day 2 of exposure was considered insignificant (i.e. <20%).
Further, mortality increased to 10, 20, 25, 30, 45 and 50% at day 10, 12, 14, 18, 19 and 20 of exposure, respectively. The reason for the observed effect is unknown and it is considered as an inadvertent mortality. F

Discussion
During a chronic study, reproduction and growth are considered to be the most sensitive endpoints. Reproduction, when calculated as a number of offspring per introduced parent, was stimulated rather than inhibited by the presence of the test item in this study. Also, no negative effect on growth of exposed daphnids was observed. However, unexpectedly high mortality rate was observed in the limit concentration. The exact reason is unknown, however, it is believed not to be caused by toxicity of the test item, because: 1) no toxic effects were observed with respect to the typically more sensitive parameters growth and reproduction, and 2) a separate acute test of Daphnia toxicity showed no mortality at the maximum water solubility concentration (loading concentration of 100 mg/L). It is possible that observed mortality was caused by mechanical effect, i.e. by undissolved test material blocking filtering apparatus of the exposed daphnids. Although there is no direct proof for
this speculation, it can be supported by the fact that a number of daphnids exposed to the test item treated solution were trapped at the surface of the test solution at the start of the test.
Therefore, the observed mortality is considered inadvertent. For this reason the NOEC for survival cannot be set based on results obtained in this study.

Experimental Conditions

The individual results are presented in:
• pH: The pH was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).
• Dissolved oxygen concentrations: The oxygen concentration complied with the requirements as laid down in the study plan (> 3 mg/L).
• Temperature: The temperatures generally complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C). The temperature measured in control vessel slightly exceeded the range (max. 23°C, .
• Total hardness: Total hardness complied with the requirements as laid down in the study plan (>140 mg CaCO3 per liter).
• The concentration of TOC in sample taken from the M7-medium was below the detection limit of the device and as such complied with the value recommended by the guideline (i.e. <2 mg TOC/L).
• The light intensity during the 16-hour daily light period was close to 400 lux, as prescribed by the study plan.
During a chronic study, reproduction and growth are considered to be the most sensitive endpoints. Reproduction, when calculated as a number of offspring per introduced parent,
was stimulated rather than inhibited by the presence of the test item in this study. Also, no

Results with reference substance (positive control):

N/A

Any other information on results incl. tables

Group Mean Cumulative Number of Juveniles per Introduced Parent and Reduction of Reproduction at the End of the Test
Test item Average exposure conc. (μg/L)	Mean	s	n	% Change^1, 2
Blank control	175.0	16.07	4
Solvent control	135.6	26.45	4
7.8	154.9	31.34	4	-14.3
1 Change in reproduction when compared to the solvent control
2 Negative numbers indicate a stimulation

Group Mean Body Lengths (mm) and Reduction of Growth of Parental Daphnids at the End of the Test
Test item Average exposure conc. (μg/L)	Mean	s	n	% Change^1, 2
Blank control	4.07	0.120	18
Solvent control	4.01	0.053	18
7.8	4.09	0.093	10	-2,1
1 Change in length when compared to the solvent control
2 Negative numbers indicate a stimulation

Exposure parameters
Condition	Range
pH	7.9-8.1
Dissolved oxygen concentration (mg O2/L)	8.3-9.2
Temperature measured in test vessels (°C)	20-21
Temperature measured continuously in control vessel (°C)	20-231
Hardness (mg CaCO3)	179-196
Total Dissolved Organic Carbon content (mg/L)	n.d.
Light intensity (Lux)
Start test	412-415
End test	400-404
n.d.: not detectable; concentration was below the detection limit of the method, 1 see study plan deviation in
Effect Parameters
Parameter	Concentration (μg/L)
NOEC for reproduction	7.8
EC10 for reproduction	>7.8*
NOEC for mortality	n.d.
NOEC for growth	7.8
* – 95% confidence intervals could not be given, n.d. – not determined

Number of Living Newborn per Replicate per Day
Nominal	Replicate	Test item - Average exposure conc. (μg/L)
day		Blank control	Solvent control	7.8
0	A	0	0	0
	B	0	0	0
	C	0	0	0
	D	0	0	0
	Mean	0	0	0
7*	A	13	9	4
	B	28	24	15
	C	26	18	27
	D	19	5	19
	Mean	22	14	16
8	A	29	91	85
	B	42	64	60
	C	28	59	41
	D	18	78	30
	Mean	29	73	54
9	A	93	1	6
	B	41	10	29
	C	42	1	3
	D	0	11	41
	Mean	44	6	20
10	A	139	197	201
	B	191	202	115
	C	184	206	149
	D	223	199	157
	Mean	184	201	156
11	A	7	21	5
	B	12	17	56
	C	10	19	12
	D	9	12	82
	Mean	10	17	39
12	A	134	2	0
	B	45	2	88
	C	97	0	0
	D	59	0	22
	Mean	84	1	28
13	A	159	105	288
	B	147	169	142
	C	157	49	160
	D	108	52	186
	Mean	143	94	194
14	A	3	33	3
	B	6	1	47
	C	1	18	4
	D	14	32	4
	Mean	6	21	15
15	A	94	0	0
	B	34	1	58
	C	58	0	9
	D	31	2	74
	Mean	54	1	35
16	A	35	96	115
	B	66	118	47
	C	56	32	51
	D	84	68	24
	Mean	60	79	59
17	A	0	0	0
	B	0	2	25
	C	0	11	0
	D	0	59	0
	Mean	0	18	6
18	A	95	0	0
	B	29	27	80
	C	74	6	19
	D	71	4	61
	Mean	67	9	40
19	A	76, 3i	171	167
	B	145	170	30
	C	107	75	41
	D	108	11	39, 5e
	Mean	109	132	69
20	A	0, 2i	0	3
	B	12	3	44
	C	0	3	0
	D	0	46	7
	Mean	3	13	14
21	A	84	0	1
	B	23	0	45
	C	85	0	32
	D	50, 1i	1	46
	Mean	61	0	31
i – number of immobile offspring, e – number of aborted eggs
* - no reproduction was observed on days 1-6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study test item did not negatively affect reproduction and growth of Daphnia magna at an average exposure concentration of 7.8 μg/L after 21 days of exposure (NOEC).

Executive summary:

The objective of the test was to evaluate the effects of test item on the mobility, the growth and the reproductive capacity of Daphnia magna. For this purpose, test organisms were exposed to aqueous solutions containing the test item at a nominal concentration of 10 μg/L. The time of the first production of young, the number of young born, immobility and other signs of intoxication observed were compared with corresponding parameters in the controls. A limit test was conducted due to the very low water solubility of test item. In addition, the nominal test concentration of 10 ug/L was selected because this was close to the previously determined water solubility limit of for test item of <8 ug/L. It should be noted that later on, the water solubility was determined to be <1.6 μg/L (Test Facility Study No. 20218324).

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the OECD guidance document number 23, 2019.

The batch of test item was a slight yellow powder with a purity of 98%. Weighing of test item and preparation of stock solutions was performed under dimmed light and stock solutions were kept in amber vessels as the test item possibly reacts with light. The test was performed in a flow-through system with target concentration of 10 μg/L. Stock solutions were prepared in Dimethyl sulfoxide (DMSO). A blank- and a solvent control were included.

The test was performed with four replicates per group, each replicate contained 5 daphnids. Exposure duration was 21 days. The condition of the parental daphnids was recorded daily.

During the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded daily. At the end of the test the length of the surviving parental daphnids was measured. Light intensity was kept as low as possible during the exposure.

Samples for analysis of exposure concentrations were taken at the start of the test and weekly thereafter. The average exposure concentration was calculated to be 7.8 μg/L.

Mortality in each of the control treatments reached 10% at the end of the exposure, hence complied with the validity criteria of the study (i.e. was ≤20%). Mortality in the limit concentration reached 50% at the end of the test. The observed mortality was considered inadvertent because: 1) no toxic effects were observed with respect to the typically more sensitive parameters growth and reproduction, and 2) a separate acute test of Daphnia toxicity showed no mortality at the maximum water solubility concentration (loading concentration of 100 mg/L).

On average 175 offspring were produced in the blank and 136 in the solvent control. Hence, the validity criterion of at least 60 offspring per parental daphnid was met.

A stimulation of reproduction was observed in the test item treated group when compared with solvent treatment (14% increase). Therefore, the NOEC for reproduction was 7.8 μg/L.

The growth of surviving daphnids (measured as the total length) was not affected by the presence of test item. Therefore, the NOEC for growth was 7.8 μg/L.

Effect parameters (μg/L) obtained in this study are summarized in the table below.

Parameter Concentration (μg/L)

NOEC for reproduction 7.8

EC10 for reproduction >7.8*

NOEC for mortality n.d.

NOEC for growth 7.8

* – 95% confidence intervals could not be given, n.d. – not determined

In conclusion, under the conditions of the present study for the test item did not negatively affect reproduction and growth of Daphnia magna at an average exposure concentration of 7.8 μg/L after 21 days of exposure (NOEC).