1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-10-28 to 2009-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

September 21, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Version / remarks:

October, 1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test material is in a white powder form.

Analytical monitoring:

yes

Details on sampling:

For analytical verification of the test item concentrations, duplicate samples containing 20 mL of the freshly prepared test media (without daphnids) were taken on day 0, 12 and day 19 from bulk preparation for all test levels and the untreated control immediately after preparation and again on days 3, 14 and 21, at the end of each exposure interval, from the pooled content of the corresponding test replicates (aged media). The samples were stored below -18°C until being transferred to laboratory where measurement occurred. All samples were analysed on content of the active substance, pencycuron by HPLC.

Vehicle:

yes

Remarks:

Acetone ( 0.1 mL/L)

Details on test solutions:

The study covered five geometrically spaced test concentrations (12.4, 24.8, 49.6, 99.2 and 198 µg a.s./L nominally = spacing factor 2.0), supplemented by an untreated dilution water (blank) control and an additional solvent control group.

Preparation of test solutions for the reported study occurred immediately before the start of each exposure-interval. A primary stock solution was prepared by dissolving a nominal amount of 100.9 mg pencycuron (tech.) in 50 mL Acetone corresponding to 1984 mg a.s./L (=stock solution A). From this solution, sub dilutions were established.

Before each following dilution step, acetoneous stock solutions were stirred by a magnetic stirrer for at least 5 minutes. From each of these stock solutions 200 µL were taken and carefully added to an amount of 2000 mL test water in order to establish concentrations of 12.4, 24.8, 49.6, 99.2 and 198 µg a.s./L. An additional solvent control was prepared by dissolving 200 µl Acetone in 2000 mL test water. All aqueous solutions were stirred for at least 19 minutes

At study initiation, the readily prepared test solutions were distributed to the corresponding replicate vessels and one <24 hours old first instar daphnid was introduced below the water surface of each test beaker. A computer generated random number sequence was used to assign the test animals impartially to the test beakers.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: waterflea (Daphnia magna)
- Age at study initiation: <24 hours old
- Stage and instar at study initiation: first instars
- Age of parental stock: 20-28 days ± 12 hours old
- Feeding during test: Food was added after introducing the test animals. Over the whole exposure period, the water fleas were fed with living cells of unicellular green algae in aqueous suspension.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS
First instars of the waterflea Daphnia magna, <24 hours old were used for this study. The culture has been maintained in 2000 mL containers (50 to 100 daphnids per container) in weekly renewed aqueous media (test medium), being placed in an climate-controlled environment under study conditions. The daphnids were fed three times per week with living cells of the green alga Desmodesmus subspicatus. The first instars used in the test are obtained by repeated screening of a breeding culture of defined age, less than 24h before test initiation. They descend from at least the fourth (or later) brood of coeval parent daphnids (20-28 days ± 12 hours old). No males, ephippia or dead animals were present in the cultures. The cultures showed no delay in first brood.

Test type:

semi-static

Water media type:

other: Elendt M7 medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

13 - 14°dH

Test temperature:

20.4 - 21.1°C

pH:

7.6 - 8.2

Dissolved oxygen:

7.2 - 9.0 mg/L

Salinity:

3 - 4°dH

Conductivity:

536 - 603 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 12.4, 24.8, 49.6, 99.2 and 198.4 µg a.s./L
Measured concentrations: 13.3, 27.7, 52.0, 102, 210 µg a.s./L (at day 21)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass beakers
- Type: closed
- Renewal rate of test solution: During the course of the reported study, all test units were refilled with freshly prepared test solutions study days 3, 5, 7, 10, 12, 14, 17 and 19 to meet the recommended renewal interval of max. 3 days.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10 replicates
- No. of vessels per control (replicates): 10 replicates
- No. of vessels per vehicle control (replicates): 10 replicates

TEST MEDIUM
For breeding of the stock culture and for testing, a fully defined, artificial water (type “Elendt M7”) was prepared as defined by the underlying OECD / EEC guidelines. The medium is prepared by dissolving mineral salts (analytical grade chemicals) in deionised water with a conductivity below 10µS/cm.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 hours light dark cycle
- Light intensity: 1200 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
Visual evaluations were performed daily throughout the whole exposure period. Behaviour of the parent females was visually evaluated by counting mobile daphnids, defined as animals with swimming movements (slight movements of antennae were not interpreted as swimming movement) within approximately 15 seconds after gentle agitation of the test vessel. Additionally possible signs on sublethal affected daphnids were recorded. Apart from parental behaviour, neonate hatch was counted and further observations on reproductive quality like sublethal effects on neonates, existence of aborted eggs and neonates mortality were performed. Therefore, initially the behaviour of freshly emerged neonates was recorded whilst remaining in the exposure solutions. Then, prior to counting, the corresponding parent daphnid was removed carefully from the beaker and placed in a quantity of the appropriate test solution. The remaining test solution containing the living offspring was then strained through a 0.2 mm screen. The neonates and possible aborted eggs were retained on the screen for counting. Counted offspring was discarded. Finally the test beakers were refilled with the test media and the parent daphnia were introduced. The onset of maturity was recorded individually for each parent female. At study termination, the body length (i.e. body length excluding the anal spine) of all surviving parent animals was determined by measurement under a scaled binocular.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

99.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: based on reduced parental body length at test termination

Details on results:

Parental body length at test termination
The mean body length of untreated control animals as given in Table 5 and Figure 1 was 4.48 mm. This body length indicated that the test animals were full grown, well-nourished females of normal body size.

Survival Rate and Sublethal Effects
No treatment related effects on parental behaviour were observable during the course of exposure.

Reproductive endpoints
The number of viable offspring was counted and recorded for each test vessel separately, performed daily from the appearance of the first offspring emergence until study termination. No offspring occurred in this study during the first week of exposure.

Neonates Behaviour and Quality of Reproductive output
Apart from the amount of living offspring, further observations on reproductive quality like sublethal effects on neonates, existence of aborted eggs and neonates mortality were performed. Observations revealed no sublethal affected offspring. So, the reported “living offspring” is identical to “total offspring”. The cumulative living offspring is given in Table 6 and Figure 2.

Average daily offspring
For each surviving parental animal the first day of reproduction was recorded. The number of living neonates per surviving parent and reproduction day was calculated by dividing the individual sum of living neonates per parent by the number of reproduction days. Counting and observation of neonates was performed daily until parent daphnids premature death or the last day of the study. The corresponding daily number of neonates for each testing group are summarised in Table 7.

Parental age at first reproduction
The onset of reproductive maturity was evaluated as possible indication for adverse effects on parental development. It is displayed as age at first offspring emergence (days) as obtained by adding the average parental age at test initiation (0.9 days) to the recorded study day of first offspring emergence (Table 8 and figure 4).

Discussion

Study quality

Sensitivity of the used daphnid breeding-strain is located within the required range as verified by periodically performed acute reference substance testing. No immobilisation of untreated control animals occurred. The reproductive output as recorded for the untreated water control group fulfilled the required minimum value of >60 neonates per female during 21 days. The average total offspring per untreated control animal revealed 132 neonates (CV=13.6%), corresponding to an average daily hatch of 9.7 neonates per untreated control female. Similar values were recorded for the solvent control group (133 neonates per parental female (CV=11,3%), corresponding to an average daily hatch of 9.6 neonates. The final body length was 4.48 mm for parental animals from untreated water control and 4.53 mm for parental animals from solvent control. These growth data indicated, that the untreated control animals were full grown, well-nourished females of normal body size. The onset of reproductive maturity was evaluated as possible indication for adverse effects on parental development was evaluated individually for each animal. The average age at first offspring emergence for parental females from pooled controls was 9.1 days (9,2 days for untreated water control, 9,0 days for solvent control), which secures a sufficient length of the breeding period as used for comparison to the breeding quality of treated animals. Since the data for all study endpoints showed no statistically significant (p=0.05, two sided) differences between both control groups, the data from both control groups were pooled and all data from treatment groups were related to the pooled control data. For water quality monitoring, temperatures, pH values and O2 concentrations, conductivity, hardness and alkalinity of the test solutions, were regularly controlled throughout the study as recommended by the underlying guidelines. As measurements show, the physical/chemical properties corresponded to the required values. Thus, the study conditions and breeding quality met the required quality criteria. Based on 3 series of weekly measurements, the actual concentrations of pencycuron were determined for samples from start and end of three representative exposure intervals. The accompanying analytical verification for freshly prepared test solutions at start of the chosen renewal intervals revealed recoveries between 104% and 120% (mean: 111%) of the corresponding nominal concentrations. The corresponding concentrations of the aged test solutions at the end of the chosen exposure intervals ranged between 99% and 115% (mean: 107%) of nominal, Since the analytically verified concentrations matched nominal values closely, all reported results are related to nominal concentrations. No contaminations of pencycuron were detected in samples from untreated water control.

 

See "Attachments" in "Overall remarks, attachments" for the tables and figures.

Validity criteria fulfilled:

yes

Conclusions:

The overall chronic NOEC for 21 days of static renewal exposure of pencycuron (tech.) to Daphnia magna, expressed as nominal test concentration, is 99.2 µg a.s./L. This NOEC based on a reduced parental body length at test termination. The corresponding LOEC is 198 µg a.s./L. Therefore the "Maximum Acceptable Toxicant Concentration" (MATC), calculated as geometric mean between NOEC and LOEC, is 140 µg a.s./L (nominally).

Executive summary:

The waterflea Daphnia magna, was exposed to Pencycuron (techn.) at nominal concentrations of 12.4, 24.8, 49.6, 99.2 and 198.4 µg a.s./L under semi-static conditions for 21 days. No mortality was observed in any control or test group. The reproduction rate was slightly reduced compared to the pooled controls at the highest test concentration, but this difference was not statistically significant at the 5% level (one-sided testing). Body length of surviving parent daphnids was statistically significantly reduced at the highest test concentration. The overall NOEC was 99.2 µg a.s./L, based on a small but significant reduction in parental body length.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-09-21 to 1991-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1330 (Daphnid Chronic Toxicity Test)

Version / remarks:

1986

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, Section 2, No. 202

Version / remarks:

April 4, 1984 - OECD Guideline for Testing of Chemicals, Section 2, No. 202, "Daphnia sp., Acute Immobilisation Test and Reproduction Test", Part II

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test material is a white solid.

Analytical monitoring:

yes

Details on sampling:

For analytical control of the test concentrations, samples were taken out of the freshly prepared test media of all test concentrations and solvent control at the first treatment period, at a treatment period in the second week (day 12) and at the last treatment period (day 19) and analysed in duplicate. Analysis was by HPLC.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: a control without any additions and a solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Other relevant information: A stock solution was prepared by dissolving the test substance in Acetone p.A. (2 g/L). The stock solution was diluted in a series of sequential dilutions with the solvent to add the same volumes of solvent (0. 1 ml/L) to each test solution. The final nominal concentrations of Pencycuron (techn.) in the test media were 12.4, 24.8, 49.6, 99.2 and 198.4 µg a.s./L (corresponding to 12.5, 25, 50, 100 and 200 µg a.s./L), a control without any additions and a solvent control (100 µl Acetone p.A./L test medium).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea (Daphnia magna Straus)
- Age at study initiation: less than 24 hours old
- Feeding during test: The Daphnia were fed with algae (Scenedesmus subspicatus)

Test type:

semi-static

Water media type:

other: Reconstituted water

Limit test:

no

Total exposure duration:

21 d

Test temperature:

18.0 - 20.0°C

pH:

7.6 - 8.7

Dissolved oxygen:

8.0 - 11.2 mg/L

Nominal and measured concentrations:

Nominal concentrations of 12.4, 24.8, 49.6, 99.2 and 198.4 µg a.s./L (corresponding to 12.5, 25, 50, 100 and 200 µg a.s./L).
Measured concentrations: 15.6, 33.7, 67.0, 125.8 and 183.1 µg a.s./L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Aeration: Before use, the test water was aerated until oxygen saturation. During the test the test media have not been aerated.
- Renewal rate of test solution (frequency/flow rate): The test medium (treated and untreated) was renewed at day 3, 5, 7, 10, 12, 14, 17 and 19 of the exposure period. By that, a total of 9 treatments were performed.
- No. of organisms per vessel: 5 animals in each glass-beaker.
- No. of vessels per concentration (replicates): 4 replicates of 5 animals in each beaker.
- No. of vessels per control (replicates): 4 replicates of 5 animals in each beaker.
- No. of vessels per vehicle control (replicates): 4 replicates of 5 animals in each beaker.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours/day
- Light intensity: 200 - 600 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Themortality of adults and the number of young were controlled three times per week before renewal of test media. Dead animals and offsprings were removed at the observation dates.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

49.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: overall nominal 21 d-NOEC

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 198.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 198.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

49.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Mortality of adults
At the end of the test the survival rate of adults of the solvent control amounted to 95%, in control to 90% (Table 1). Up to the test concentration of nominal 49.6 µg a.s./L the survival rate was 80% or higher. First in the nominal concentration of 99.2 µg a.s./L the mortality significantly increased (35%), in the highest concentration tested (198.4 µg a.s./L) 55% of the initial number of Daphnia were dead after the exposure time of 21 days.

Reproduction rate
Table 2 shows the total number of alive off-springs (cumulative values) reproduced by all adults within 21 days of continuous exposure to various concentrations of Pencycuron (techn.). The first young Daphnia released from their parent animals were observed in the controls and in all test concentrations on day 10. The reproduction rate calculated for each individual surviving Daphnia is given in Table 3. The reproduction rate of Daphnia in the solvent control was 105.5 ± 25.0 (mean ± SD) alive off-springs per adult. Obviously no toxic effect of the test substance on the reproduction rate was observed up to the highest concentration of 198.4 µg a.s./L. The exposed Daphnia, which survived until the end of the test, had no significantly* different reproduction rate than Daphnia in controls (see Table 3).

Body length of adult Daphnia
No significant influence of the test substance on the body length of surviving adult Daphnia was observed. The differences between the mean body length of Daphnia (measured at the end of the test) were low, a clear concentration-effect-relation could not be determined (Table 4).

Signs of intoxications
With exception of the reported mortality, no particular signs of intoxications were observed at the test animals during the test duration.

The 21d-NOEC values are =198.4, =198.4, and 49.6. µg a.s./L for growth, reproduction and survival, respectively, and an overall nominal 21d-NOEC of 49.6 µg a.s./L.

* calculated by the DUNNETT-Test, two-sided, p=0.05

See "Attachments" in "Overall remarks, attachments" for the tables.

Validity criteria fulfilled:

yes

Conclusions:

Measured concentrations of Pencycuron were high at all concentrations except the highest concentration of 200 µg a.s./L. At 49.6 µg a.s./L (21d-NOEC for survival) the measured values were 114.6, 182.2 and 108.2 of nominal. Concerning the reproduction rate and the body length of surviving Daphnia, no toxic effect was observed up to the highest concentration tested of 183.1 µg a.s./L (measured concentration, nominal 198.4 µg a.s./L). The 21d-NOEC values are =198.4, =198.4, and 49.6. µg a.s./L for growth, reproduction and survival, respectively. An overall nominal 21d-NOEC of 49.6 µg a.s./L was obtained.

Executive summary:

The cladoceran, Daphnia magna, was exposed to Pencycuron (techn.) at nominal concentrations of 12.4, 24.8, 49.6, 99.2 and 198.4 μg a.s./L (corresponding to 12.5, 25, 50, 100 and 200 μg a.s./L) under semi-static conditions for 21 days. Concerning the reproduction rate and the body length of surviving Daphnia, no toxic effect was observed up to the highest concentration tested of 183.1 µg a.s./L (measured concentration, nominal 198.4 µg a.s./L). The 21d-NOEC values are ≥198.4, ≥198.4, and 49.6. µg a.s./L for growth, reproduction and survival, respectively. An overall nominal 21d-NOEC of 49.6 µg a.s./L was obtained.

Description of key information

A study was conducted to investigate the chronic toxicity of Pencycuron to Daphnia magna over 21 days. Based on nominal concentrations of Pencycuron the 21-d NOEC was determined to be 0.0496 mg/L.

 

Test species	Result	Assessment	Reference
Daphnia magna	21-d NOEC = 0.0496 mg a.s./L (nominal)	Key study	Memmert (1991)
Daphnia magna	21-d NOEC = 0.0992 mg a.s./L (nominal)	Supporting study	Bruns (2009)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.05 mg/L