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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-09 to 2012-07-14
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:
yes
Remarks:
River water was used instead of standard water
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: Control - 0.0855 – 0.188 – 0.413 – 0.909 – 2.00 mg/L
- Sampling method: Various concentration levels and the control were analytically verified twice via LC-MS/MS from freshly prepared and old media (D0/D1 and D2/D3).
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1.00 g/L was prepared with demineralised water 24 h prior to start of exposure and prior to each water renewal, respectively. The pH-value of the demineralised water was adjusted to pH 2 ± 0.1 by adding 2 M HCl. Stock solution preparations were performed each day, 24h before use. The stock solution was stirred with a magnetic stirrer for 24 h at approximately 50 °C in a laboratory incubator. Thereafter the stock solution was cooled down to ambient temperature. The test concentrations were prepared from this stock solution by dilution without further adjustment of the pH-value.
- Eluate: Natural river water
- Nominal concentrations: 0.0855 – 0.188 – 0.413 – 0.909 – 2.00 mg/L
- Controls: 7 fish in dilution water without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.37 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.233 g
- Method of breeding: Holding was performed at the test facility at 23+/-2 °C and diffuse light (0.1 - 10 µmol photons.m-2.s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test

ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
daily renewal
Hardness:
Total Hardness River Water: 271 mg/L
Test temperature:
Please refer to "Any other information on materials and methods"
pH:
Please refer to "Any other information on materials and methods"
Dissolved oxygen:
Please refer to "Any other information on materials and methods"
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal (after 0, 24, 48, 72 hours)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water Parameters of the River Water
River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2012-03-20
Weather on Day of Sampling Sunny, slightly clouded; approx. 15°C
Colour Yellowish to brownish, clear
pH 7.93
Conductivity [µS/cm] 856
Dissolved Oxygen [mg O2/L] 11.2
DOC [mg C/L] 2.80*
TOC [mg C/L] 3.66*
Ammonium-N [mg N/L] 0.07
Nitrate-N [mg N/L] 3.40
Total Nitrogen [mg N/L] 3.80
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] < 0.20
Suspended Matter [mg/L] 14.0
Total Hardness [mg CO3/L] 271
Storage conditions 15 ± 2°C
*measured with NPOC-method

- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality [%] in the Preliminary Test
(n = 5)
Nominal test item
concentration
[mg/L] Test Duration [hours]
24 48 72 96
10 0 100 100 100
1.0 0 0 40 100
0.1 0 0 0 0
Control 0 0 0 0
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.95 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: (0.909 - 2.0)
Details on results:
- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item showed turbidity in the tested loading level during exposure.
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
Initially, the study report reported arithmetic mean of the four measured concentrations per test group. Since, more than 20% of the test item was lost during each 24 hour exposure period, the mean concentration per 24h exposure period should have expressed as the geometrical mean between fresh and old media concentrations. Then, the mean measured concentration should have been based on the arithmetic mean of the two geometrical means. The LC50 was revisited using Toxrat 3.2. The value did not change much (less than 10% difference). The pdf is attached to this RSS.

Sublethal observations / clinical signs:

Observations in the Test Vessels

Nominal test item concentration

Mean measured test item concentration

Effect *

Test duration [hours]

[mg/L]

[mg/L]

24

48

72

96

2.00

1.48

(E)

-

-

7/7

---

(2.9)

-

7/7

-

---

(2.8)

5/7

-

-

---

(2.7)

7/7

-

-

---

(2.6)

7/7

7/7

-

---

(2.5)

-

4/7

-

---

(2.4)

-

3/7

-

---

0.909

0.607

(2.6)

-

-

3/7

3/7

(1)

7/7

7/7

4/7

4/7

0.413

0.264

(1)

7/7

7/7

7/7

7/7

0.188

0.126

(1)

7/7

7/7

7/7

7/7

0.0855

0.058

(1)

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

Cumulative Mortality [%] in the Test Vessels

Nominal
 test item concentration

Mean measured
 test item concentration

Test duration [hours]

[mg/L]

[mg/L]

24

48

72

96

2.00

1.48

0

0

100

100

0.909

0.607

0

0

0

0

0.413

0.264

0

0

0

0

0.188

0.126

0

0

0

0

0.0855

0.058

0

0

0

0

Control

Control

0

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
In this study N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] was found to cause lethal effects to Zebrafish after 96 hours at a nominal concentration of 2.00 mg/L. The nominal LC50 (96 hours) was 1.35 mg/L, corresponding to a mean measured LC50 of 0.95 mg/L. All effect levels are given based on the nominal and mean measured concentration levels of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate].
Executive summary:

The acute toxicity of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] in natural river water to fish (zebrafish), was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2012-07-09 to 2012-07-14, at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test with daily renewal of the test media was conducted with the nominal test item concentrations of 0.0855 – 0.188 – 0.413 – 0.909 – 2.00 mg/L (spacing factor 2.2), corresponding to mean measured concentrations of 0.058 – 0.126 – 0.264 – 0.607 – 1.48 mg/L. Duration of the test was 96 hours. 7 test organisms were exposed to each test concentration and control. Water quality parameters temperature, pH-value and O2-saturation measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The determination of various concentrations of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] and the control was carried out via LC-MS/MS from freshly prepared media after 0 and 72 h, and from corresponding 24 h old test media after 24 and 96 h. Sorption of the test item to the glassware of 24 hours aged test media was determined in the aquarium of the nominal concentration 0.909 mg/L.

The percent of nominal of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate]in freshly prepared media were in the range of 98 – 110 % and in 24 h aged test media were in the range of 36 - 55 % of the nominal values, respectively. All effect levels are given based on the nominal and the mean measured concentrations of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate].

The aquatic ecotoxicity tests with N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] were performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PECaquatic,bulk is compared with the PNECaquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible.

The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PECbulk clearly provide a more appropriate assessment of risks for the environment. An individual test design was applied. Natural river water was used as dilution medium. 96h-LC50 was 1.35 mg/L.

Description of key information

The 96h-LC50 is 1.34 mg/L in a OECD guideline 203 test in natural river water with Danio rerio under GLP (reliability K1).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
1.35 mg/L

Additional information

The acute toxicity of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] towards Danio rerio in natural river water was determined according to OECD guideline 203 under GLP. A semi-static test with daily renewal of the test media was conducted with the nominal test item concentrations of 0.0855 – 0.188 – 0.413 – 0.909 – 2.00 mg/L (factor 2.2), corresponding to mean measured concentrations of 0.0625 – 0.140 – 0.290 – 0.668 – 1.56 mg/L. Duration of the test was 96 hours. The 96h-LC50 based on nominal concentrations was found to be 1.35 mg/L.