N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

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:

June 3, 2013 to June 7, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals, No.203, July 17, 1992, “Fish, Acute Toxicity Test”

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Japanese Test Guidelines

Version / remarks:

“Fish acute toxicity test” stipulated in the "Test Methods of the New Chemical Substances etc." (March 31, 2011, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, partly amended by Yakushokuhatsu 0402 No.1, Heisei 24.03.28 Seikyoku No.2, Kanpokihatsu No. 120402001 on April 2, 2012)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Frequency of measurement: At the start and the end of exposure.
Sampling methods: Samples were taken from the middle depth of the test vessel.
Sampling amount: Approximately 10 mL was sampled for all test groups.

Vehicle:

no

Details on test solutions:

The provided substance was weighed and mixed with the dilution water in a vessel for preparation.
Then this mixture was stirred by a magnet stirrer for 48 hours in order to prepare 100 mg/L of the test solution.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Species: Killifish (Oryzias latipes)
Reason for selection of the species: Use of the species is recommended in the test guidelines.
Supply source: Kurume Laboratory, Chemicals Evaluation and Research Institute (in-house production)
Body length: Entire length 2.3 ± 1.2cm
Group allocation: Random sampling

Acclimatization:
Date of hatching: November 14, 2012 (age in month when used for the test: 6 months)
Rearing water: Dechlorinated tap water
Acclimatization method: Flow through system
Dissolved oxygen: 80% and above of the saturated dissolved oxygen concentration
Water temperature: 24 ± 1 °C
Lighting cycle: Room light, 16-hour light/8-hour dark cycle
Feeding: Formulated food, crumble type 2C, for juvenile corps (Nippon Formula Feed Manufacturing Company Limited)
Frequency and amount of feed: Feeding everyday by 3% amount of fish weight/day
Medication bath: None
Acclimatization period: 42 days (from April 22, 2013 to June 3, 2013)
Mortality of fish used for the test: 0% (during 7 days before used for the test)
No feeding period: Fish was not fed for 24 hours before used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified in the study report

Hardness:

Not specified

Test temperature:

Water temperature of the test solution was 24.2 to 24.7 °C.

pH:

pH was 7.8 to 7.9

Dissolved oxygen:

The dissolved oxygen concentration was 8.0 to 8.2 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

100 mg/L nominal (as a limit test at the upper concentration limit for used test method)

Details on test conditions:

Test equipment and apparatus
Test vessel: 3 L glass vessel (diameter 16cm, depth 16cm)
Cover: A transparent cover made of plastic
Thermostatic bath: Plastic water bath, heating/cooling unit HCA 250 (Sato Kogei Co., Ltd.)

Test conditions
Exposure system: Static water system (without exchange of the test water)
Exposure period: 96 hours
Test concentration: 100 mg/L (as a limit test at the upper concentration limit for used test method)
This test concentration was determined based on the result of the preliminary study.
Control group: Dilution water without the test substance, which was treated by the same preparation method for the test solution.
Number of the replicates: 1 replicate/test group
Number of test organisms: 7 organisms/test group
Volume of the test solution: Approximately 2.8 L/test group
Water temperature: 24 ± 1 °C
Aeration: Moderate aeration
pH adjustment: None
Lighting: Room light, 16-hour light/8-hour dark cycle
Feeding: Food was not given.

Observation and measurement
Observation of the test organisms
Fish were inspected for their death and general symptoms at 3, 24, 48, 72 and 96 hours after the start of exposure. The individual was considered as dead if there was no visible movement (movement of proboscis or gill cover etc.,) and if gently touching of the caudal peduncle with a glass rod produced no reaction.

Body length and weight of the test organisms
Body lengths and weights of the test organisms of the control group were measured at the end of exposure.

Condition of the test solution
Condition of the test solution was observed at the start and the end of exposure.

Quality of the test solution
Measurement items: Concentration of dissolved oxygen concentration, pH and water temperature
Frequency of measurement: At the start of exposure and following every 24 hours
Measurement methods: Aliquot of the test solution sampled from the test vessel was measured.
Instruments: Dissolved oxygen meter, YSI MODEL 58 (YSI Incorporated), Portable pH meter, HM-21P (TOA D.K.K), Certified glass stem thermometer

Test substance concentration in the test solution
Frequency of measurement: At the start and the end of exposure.
Sampling methods: Samples were taken from the middle depth of the test vessel.
Sampling amount: Approximately 10 mL was sampled for all test groups.

Reference substance (positive control):

yes

Remarks:

copper sulfate (II) pentahydrate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality
During the exposure period, death of the organism was not observed in the test concentration group. In addition, death of the organism was not observed in the control group, and the validity criteria (number of dead fish should not exceed 1) was fulfilled.

Observation results of symptom
No symptom of test organisms was observed in the control group. All these observation results were based on comparison with the control group, and no symptom of the test organisms in the test concentration group was observed during the exposure period.

The size of the test organisms
[Mean value ± standard deviation (n = 7)]
Body length 2.4 ± 0.1 cm
Body weight 0.10 ± 0.02 g

Observation and measurement results of the test solution
Condition of the test solution
The test solution was colorless and transparent at the start of the exposure and the condition was the same at the end of exposure.

Water quality of the test solution
The dissolved oxygen concentration was 8.0 to 8.2 mg/L, the pH was 7.8 to 7.9, and water temperature of the test solution was 24.2 to 24.7 °C. The dissolved oxygen concentration fulfilled the validity criteria (at 60% or above of the saturated dissolved oxygen concentration* at the test water temperature).
* Saturated dissolved oxygen concentrations at 23 to 25oC are 8.39 to 8.11 mg/L, JIS K 0102: 2008

Test substance concentration in the test solution
The measured concentrations of the test substance in test solution were 100 mg/L both at the start of exposure and at the end of exposure (100 % of the nominal concentration). These concentrations were maintained within ± 20 % of the nominal concentration.

Results with reference substance (positive control):

Acute toxicity tests are periodically performed with the reference substance. The latest data is shown below.
Reference substance: copper sulfate (II) pentahydrate (special grade, Lot No. LAJ5671, Wako Pure Chemical Industries, Ltd.)
Test period: from May 13, 2013 to May 17, 2013
96-hour LC50: 0.45 mg/L
This value was within the determined range of the background data in Kurume Laboratory, which is a range of “mean ± 2 x standard deviation” [mean ± standard deviation: 0.53 ± 0.19 mg/L (n = 52)]. All these data are expressed as amount of Copper sulfate (II).

Reported statistics and error estimates:

Not specified

Sublethal observations / clinical signs:

Cumulative mortality

Nominal concentration (mg/L)	Cumulative mortality (%)
	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0	0
100	0	0	0	0	0

 

Observed abnormal response

Nominal concentration (mg/L)	Result of observation (Left column: Number of affected fish/Total survival number Right column: Symptom detail)
	3 hours		24 hours		48 hours		72 hours		96 hours
Control	0/7	N	0/7	N	0/7	N	0/7	N	0/7	N
100	0/7	N	0/7	N	0/7	N	0/7	N	0/7	N

N: Normal (No abnormal response)

 

Dissolved oxygen concentration of test solutions

Nominal concentration (mg/L)	At the start	24 hours	48 hours	72 hours	At the end
Control	8.2	8.2	8.2	8.0	8.2
100	8.2	8.2	8.2	8.1	8.2

Unit: mg/L

 

pH of test solutions

Nominal concentration (mg/L)	At the start	24 hours	48 hours	72 hours	At the end
Control	7.8	7.8	7.8	7.9	7.8
100	7.8	7.8	7.8	7.9	7.8

 

Temperature of test solutions

Nominal concentration (mg/L)	At the start	24 hours	48 hours	72 hours	At the end
Control	24.3	24.7	24.5	24.5	24.2
100	24.3	24.7	24.5	24.5	24.3

Unit: °C

 

Measured concentrations of test item in test solutions

Nominal concentration (mg/L)	Measured concentration (mg/L) (Percentage of measured concentration versus nominal concentration %)
	At the start	At the end	Geometric mean
Control	n.d.	n.d.
100	100 (100)	100 (100)	100 (100)

n.d.: <20.0 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The test was carried out as a limit test to confirm the presence or absence of the effect of the test substance to the test organism at the upper concentration limit (100 mg/L) specified in used test method. As a result, there is no effect to the test organisms and therefore it is considered that the test substance causes no acute effect to the test organisms at the upper concentration limit of the test method. The test substance concentration in the test solution was maintained ±20% of the nominal concentration, and the environmental conditions were within the proper range for the test. Therefore, it was considered that this study was in accordance with the test method.

Both 48 and 96-hour LC50 of the test substance to killifish were > 100 mg/L.

Executive summary:

Test organism: Killifish (Oryzias latipes)

Dilution water: Dechlorinated tap water

Test groups: 100 mg/L group and a control group

Preparation of test solutions: Prepared by dissolving the test substance in the dilution water and then stirring for 48hours.

Exposure system: Static water system

Exposure period: 96 hours

Number of replicates: 1 replicate per test group

Number of test organisms: 7 organisms per test group

Volume of the test solution: Approximately 2.8L per test group

Water temperature: 24.2 - 24.7 °C

Aeration: Moderate aeration

Lighting: Room light, 16-hour light/8-hour dark cycle

Feeding: None

Measurement of the test substance concentration: HPLC method (at the start and end of exposure)

 

The test was carried out as a limit test to confirm the presence or absence of the effect of the test substance to the test organism at the upper concentration limit (100 mg/L) specified in used test method. As a result, there is no effect to the test organisms and therefore it is considered that the test substance causes no acute effect to the test organisms at the upper concentration limit of the test method. The test substance concentration in the test solution was maintained ±20% of the nominal concentration, and the environmental conditions were within the proper range for the test. Therefore, it was considered that this study was in accordance with the test method.

 

Both 48 and 96-hour LC50 of the test substance to killifish were > 100 mg/L.

Description of key information

96-hour LC50 of the test substance to killifish: > 100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The test was carried out as a limit test to confirm the presence or absence of the effect of the test substance to the test organism at the upper concentration limit (100 mg/L) specified in used test method. As a result, there is no effect to the test organisms and therefore it is considered that the test substance causes no acute effect to the test organisms at the upper concentration limit of the test method.

Both 48 and 96-hour LC50 of the test substance to killifish were > 100 mg/L.