N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2018 to 27 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: White crystalline powder
Batch: AAN0680
Purity/Composition: 99.9 %
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: 30 August 2018 (expiry date)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, samples were taken from the highest test concentration after 48 hours of exposure when all fish had died. The method of analysis is described in the appended Analytical Report.
Frequency at t=0 h and t=96 h
Volume 0.8 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Additionally, reserve samples of 0.8 mL were taken from all test solutions 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 on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride tested was a white crystalline powder with a purity of 99.9% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length Range-finding test: 2.5 ± 0.1 cm
Final test: 3.0 ± 0.2 cm
Mean weight1 Range-finding test: 0.17 ± 0.03 g
Final test: 0.28 ± 0.06 g
Characteristics F1 from a single parent-pair bred in UV-treated water.
Reason for selection This system has been selected as an internationally accepted species.
Total fish used 54
Quarantine/Acclimatisation: At least 12 days after delivery
Medium Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Measurements Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters Were kept within the optimum limits for the respective fish species.
Feeding Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3 per litre

Test temperature:

20-24°C

pH:

7.7 ± 0.3

Dissolved oxygen:

oxygen > 60% of air saturation

Nominal and measured concentrations:

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.13, 0.90, 0.86, 3.7 and 11 mg/L at the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
Species Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length Range-finding test: 2.5 ± 0.1 cm
Final test: 3.0 ± 0.2 cm
Mean weight1 Range-finding test: 0.17 ± 0.03 g
Final test: 0.28 ± 0.06 g
Characteristics F1 from a single parent-pair bred in UV-treated water.
Reason for selection This system has been selected as an internationally accepted species.
Total fish used 54
Quarantine/Acclimatisation At least 12 days after delivery
Medium Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Measurements Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters Were kept within the optimum limits for the respective fish species.
Feeding Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.


OTHER TEST CONDITIONS
Test duration 96 hours
Test type Static
Test vessels 6.5 litres, all-glass, containing 5 litres of test solution
Test medium A djusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish 7 per concentration
Loading 0.39 g fish/Litre, i.e. 7 fish per 5 litres of test medium
Illumination 16 hours photoperiod daily
Aeration Aeration was introduced after 2 days of exposure and maintained until the end of the test
Feeding No feeding from 24 hours prior to the test and during the total test period
Introduction of fish Within 25 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and other effects At 2½, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.
Dissolved oxygen content, pH and temperature Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

TEST CONCENTRATIONS
Test item 10, 18, 32, 56 and 100 mg/L
Control Test medium without test item or other additives

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

based on average exposure concentrations (95% confidence interval between 3.8 and 5.6 mg/L)

Basis for effect:

mortality (fish)

Details on results:

Range-Finding test
All fish exposed to the highest nominal concentration of 100 mg/L died within 48 hours of exposure. No mortality or clinical effects were observed at any of the lower nominal test concentrations during the 96h test period. The expected LC50 was between nominal concentrations of 10 and 100 mg/L.
Based on these results samples taken from nominal 10 and 100 mg/L were analysed. Both the scheduled and the reserve samples were measured, because the first set of samples (the scheduled samples) showed a deviating low concentration at nominal 10 mg/L at the start of the test. The initial concentrations, based on the results of the reserve samples, were 0.040 and 5.3 mg/L, respectively. These concentrations seemed to remain stable during the test period although the recovery of the test item concentrations at nominal 10 mg/L after 24 and 96 hours of exposure was relatively high (309-337% of initial). Recovery of test item concentrations at nominal 100 mg/L was 93-115% of initial. See also Table 3 of the appended Analytical Report.
All test conditions were maintained within the limits prescribed by the study plan.

Measured Concentrations
Analysis of the samples taken at the start of the final test showed measured concentrations of 0.13, 0.90, 0.86, 3.7 and 11 mg/L at the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively. Concentrations up to and including nominally 56 mg/L seemed to remain stable during the test period although the recovery of the test item concentrations at nominal 10 and 32 mg/L after 96 hours of exposure were relatively high (530 and 150% of initial, respectively). The recovery of test item concentration at nominal 100 mg/L was 35% of initial after 48 hours of exposure. This could be related to the fact that the initial concentration of 11 mg/L was significantly higher than in the range-finding test.
Based on these results, the average exposure concentrations were calculated, and used for the determination of the LC50 values.

Mortality and Other Effects
At the highest average exposure concentration of 6.5 mg/L all fish died within 48 hours of exposure. One of these fish showed loss of equilibrium after 24 hours of exposure. At the end of the 96 hour test period one fish exposed to an average exposure concentration of 3.9 mg/L had died. No fish exposed to any of the lower concentrations and the control had died or showed clinical effects during the entire exposure period of 96 hours. The responses recorded in this test allowed for reliable determination of an LC50.

Results with reference substance (positive control):

PCP
Nominal conc. (mg/L) Initial No of fish C umulative number of dead fish recorded at various time points after start of exposure Total Mortality (%)
2½h 24h 48h 72h 96h
0.10 5 0 0 0 0 0 0
0.22 5 0 5 5 5 5 100
0.46 5 0 5 5 5 5 100

Reported statistics and error estimates:

The 24h-LC50 could not be determined because the observed effects were below 50%.

The 48 and 72h-LC50 could not be determined using the maximum likelihood estimation method. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

The 96h-LC50-value was calculated from the probits of the percentages of dead fish and the logarithms of the corresponding average test item concentrations using the maximum likelihood estimation method.

ToxRat Professional v 3.2 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion,the 96h-LC50 was 4.6 mg/L based on average exposure concentrations (95% confidence interval between 3.8 and 5.6 mg/L).

Executive summary:

The objective of the study was to evaluate 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for its ability to generate acute toxic effects inCyprinus carpioduring an exposure period of 96 hours and, if possible, to determine the LC₅₀at all observation times.

The study procedure described in this report was based onthe OECD guideline No. 203, 1992.

The Safety Data Sheet, as provided by the sponsor, stated that 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is soluble in water but may decompose. During the development and validation of the analytical method it was observed that the recovery of Quality Control (QC) samples prepared in ISO-medium was better than the recovery of QC samples in double distilled water (either at pH 7 or pH 9), i.e. recovery of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloridein ISO medium was ca. 60% while recovery in double distilled water was ca. 30% (Charles River Den Bosch Project 519080). Therefore, it was decided to measure the parental compound during the fish study.

A final test was performed based on the results of a preceding range-finding test.

 

Seven fish per group were exposed to a control and to nominal concentrations of 10, 18, 32, 56 and 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken at the start of the test and after all fish had died in a group or otherwise at the end of the test.

 

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.13, 0.90, 0.86, 3.7 and 11 mg/L at the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively. Concentrations up to and including nominally 56 mg/L seemed to remain stable during the test period although the recovery of the test item concentrations at nominal 10 and 32 mg/L after 96 hours of exposure were relatively high (530 and 150% of initial, respectively). The recovery of test item concentration at nominal 100 mg/L was 35% of initial after 48 hours of exposure. This could be related to the fact that the initial concentration of 11 mg/L was significantly higher than in the range-finding test. Based on these results, the average exposure concentrations were calculated to be 0.30, 0.97, 1.1, 3.9 and 6.5 mg/L. The average concentrations were used for the determination of the LC₅₀values.

A relatively steep dose-response curve was obtained between average concentrations of 1.1 and 6.5 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion,the 96h-LC₅₀was 4.6 mg/L based on average exposure concentrations (95% confidence interval between 3.8 and 5.6 mg/L).

Description of key information

Seven fish per group were exposed to a control and to nominal concentrations of 10, 18, 32, 56 and 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken at the start of the test and after all fish had died in a group or otherwise at the end of the test. The study procedure described in this report was based onthe OECD guideline No. 203, 1992.

 

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.13, 0.90, 0.86, 3.7 and 11 mg/L at the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively. Concentrations up to and including nominally 56 mg/L seemed to remain stable during the test period although the recovery of the test item concentrations at nominal 10 and 32 mg/L after 96 hours of exposure were relatively high (530 and 150% of initial, respectively). The recovery of test item concentration at nominal 100 mg/L was 35% of initial after 48 hours of exposure. This could be related to the fact that the initial concentration of 11 mg/L was significantly higher than in the range-finding test. Based on these results, the average exposure concentrations were calculated to be 0.30, 0.97, 1.1, 3.9 and 6.5 mg/L. The average concentrations were used for the determination of the LC₅₀values.

A relatively steep dose-response curve was obtained between average concentrations of 1.1 and 6.5 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.5 mg/L