Naphthenic acids, reaction products with diethylenetriamine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-23 to 2012-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

and Council Regulation (EC) No. 440/2008 Method C.1

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, reaction products with diethylenetriamine

EC Number:

268-610-1

EC Name:

Naphthenic acids, reaction products with diethylenetriamine

Cas Number:

68131-13-5

Molecular formula:

not applicable

IUPAC Name:

Naphthenic acids, reaction products with diethylenetriamine

Details on test material:

- Name of test material (as cited in study report): Napthenic acids, reaction products with diethylenetriamine
- Physical state: Liquid, brown viscous
- Analytical purity: 100 % (UVCB)
- Purity test date: 2011-05-26
- Lot/batch No.: ESD0011831
- Expiration date of the lot/batch: 04/2013
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.625 – 1.25 – 2.50 – 5.00 – 10.0 mg/L
- Sampling method:
Analytical evaluation of the various concentrations of Naphthenic acids, reaction products with diethylenetriamine and the control was carried out via LC-MS/MS from freshly prepared media after 0 and 48 h and from corresponding aged test media after 48 and 96 h or whenever 100 % mortality occurred. The recovery rates should be within ± 20 % of the nominal or measured initial values. Due to the test item properties, the nominal concentrations were used for calculation of the effect values.
- Sample storage conditions before analysis:
All samples were stored at room temperature until start of analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 g/L was prepared with methanol in a 10 mL measuring flask (1000 mg test item were weighed out). Further stock solutions were prepared out of this stock solution by diluting with methanol. Therefore, appropriate aliquots of the stock solution of 100 g/L were pipetted directly into measuring glass flasks (capacity 5 mL) and filled up to
5 mL with methanol.
The measuring glass flasks, containing the stock solutions prepared with methanol, were closed with a glass stopper and opened only for taking aliquots for preparation of stock solutions and test concentrations, respectively. After using these solutions, they were stored at 6 ± 2 °C.
- Eluate: Natural River Water
- Differential loading: 0.625 – 1.25 – 2.50 – 5.00 – 10.0 mg/L ; Methanol: 0.1 mL/L test medium
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

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: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.50 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.23 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Total Hardness at day 0: 278 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:
River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2011-09-15
Weather on Day of Sampling Sunny, slightly clouded; approx. 18°C
Colour Yellowish to brownish, clear
pH 8.02
Conductivity [µS/cm] 960
Dissolved Oxygen [mg O2/L] 9.23
NPOC [mg C/L] 3.34
Ammonium-N [mg N/L] 0.10
Nitrate-N [mg N/L] 2.50
Total Nitrogen [mg N/L] 3.70
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] 0.23
Suspended Matter [mg/L] 15.6
Total Hardness [mg CO3/L] 278
Storage conditions 15 ± 2°C

- Culture medium different from test medium: yes
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured
measurements 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
Nominal test item concentration
[mg/L] Test Duration [hours]
24 48 72 96
10 100 100 100 100
1 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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:

LC50-values and corresponding confidence intervals after 24 – 48 hours exposure, were calculated by 0 to 100 variable slope regression. LC10,20-values could not be estimated since only one single concentration level gave partial response.
After 72 – 96 hours only 0 and 100 % mortality occurred. Hence the LC50 after 72 – 96 hours is the geometric mean value of the nominal concentrations with 0 and 100 % mortality. The concentrations causing 0 and 100 % were chosen as lower and upper confidence limit, respectively. Therefore LC10,20-values were not applicable and were given as a range of those concentrations.
The concentrations leading to 0 and 100% mortality (LC0 and LC100) as well as the no effect concentration (NOEC) after 96 h were determined directly from the test results.
The chosen methods for determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on nominal concentrations of Naphthenic acids, reaction products with diethylenetriamine. The test item Naphthenic acids, reaction products with diethylenetriamine was found to be toxic to zebrafish after 96 h. At the nominal test item concentration of 0.625 mg/L no mortality or non lethal effects were observed. Therefore the NOEC was laid down as 0.625 mg/L. The LC50 after 96 hours is 3.54 (2.50 – 5.00) mg/L.

Executive summary:

The acute toxicity of the test item Naphthenic acids, reaction products with diethylenetriamine(batch no.: ESD0011831)to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2012-07-23 to 2012-07-27 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany .
A semi-static test with renewal of the test media after 48 h was performed with the nominal concentrations of 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L. Methanol was used as solvent for preparation of the test concentrations. Duration of the test was 96 hours. 7 test organisms were exposed to each concentration level, the solvent control and the control. Water quality parameters pH-value, temperature and oxygen-saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

Various concentrations of the test item and the control were analytically verified via LC-MS/MSfrom freshly prepared media after 0 and 48 h and from aged media after 48 and 96 h or whenever 100 % mortality occurred. Four precursor ions representative for the present mass ranges were chosen for the analysis. The measured concentrations for Naphthenic acids, reaction products with diethylenetriaminein freshly prepared media were in the range of 93 – 130 % of the nominal values. In the old media the test item gave recoveries of 64 – 96 % of the nominal values. Adsorption of the test item to the test vessels in the old media after 48 and 96 h of exposure was determined from all concentration levels. The adsorbed amount of the test item was in the range of 9 to 22 % of the applied amount.

The test item Naphthenic acids, reaction products with diethylenetriamine is a cationic substance, which potentially sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results deriving from such tests depend on the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon to which the test item can sorb partially reduces the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the test substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). This so called Bulk Approach is described by ECETOC (2003). Due to the properties of the test item nominal concentrations were used instead of measured ones

NOEC and LC-Values with 95 % Confidence Intervals (0 – 96 hours)

                   Based on nominal test item concentrations [mg/L]

Test duration [hours]	LC10 (p = 95 %)	LC20 (p = 95 %)	LC50 (p = 95 %)
24	2.50^1) LC10 5.00^2)	2.50^1) LC10 5.00^2)	5.13 (2.50^1)  – 10.0^4))
48	2.50^1) LC10 5.00^3)	2.50^1) LC10 5.00^3)	5.04 (2.50^1)  – 10.0^4))
72	2.50^1) LC10 5.00^4)	2.50^1) LC20 5.00^4)	3.54 (2.50^1)  – 5.00^4))
96	2.50^1) LC10 5.00^4)	2.50^1) LC20 5.00^4)	3.54 (2.50^1)  – 5.00^4))
LC0 = Highest test item concentration with 0 % mortality after 96 hours	2.50
LC100 = Lowest test item concentration with 100 % mortality after 96 hours	5.00
NOEC (0 – 96 h) = No observed effect concentration	0.625