Naphthenic acids, reaction products with diethylenetriamine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-18 to 2012-07-24

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 202 (Daphnia sp. Acute Immobilisation Test)

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: 0.250 - 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L
- Sampling method:
All concentration levels, the solvent control and the control were analytically verified by LC-MS/MS in the fresh media (0 h) and old media (48 h). Samples of the old media were taken directly from the test vessels containing daphnids.
For the quantification of sorption to the glassware, additional replicates without daphnids were prepared for the concentration levels of 1.00, 2.00 and 4.00 mg/L and incubated under test conditions. Sorption to the walls of the test vessel was checked as specified below.
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:
For each test concentration a separate stock solution was prepared in methanol (solvent) as described below:
A stock solution of 80.0 g/L was prepared in a measuring flask (50 mL capacity) by directly weighing of 4000 mg test item into the measuring glass flask and filling up with methanol to 50 mL thereafter.
Further stock solutions were prepared out of this stock solution by diluting with methanol. Therefore, appropriate aliquots of the stock solution of 80.0 g/L (as specified in the table below) were taken and pipetted directly into measuring glass flasks (10 mL capacity) and filled up to 10 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 / test concentrations. Thereafter they were stored at 6 ± 2 °C. The test concentrations were freshly prepared at the beginning of the exposure phase (0 h) by adding 0.1 mL stock solution to 1 L of dilution water. The test concentrations were homogenised by agitation before used in the test.
- Eluate: Natural river water
- Differential loading: 0.250 - 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L
- Controls: Four replictes of dilution water without test item tested under the same consitions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 ml/L

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 278 (mg CaCO3/L)

Test temperature:

19 - 20 °C.

pH:

Water Quality Parameters at 0 h (Fresh Media)
(measured in one additional replicate per concentration level, solvent control and control)
Nominal
Test Item Concentration
[mg/L] pH-Value
8.00 8.49
4.00 8.49
2.00 8.51
1.00 8.50
0.500 8.51
0.250 8.50
Solvent Control 8.47
Control 8.41

Water Quality Parameters after 48 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
8.00 7.59 7.64 7.53 7.53
4.00 7.63 7.60 7.58 7.56
2.00 7.70 7.74 7.70 7.68
1.00 7.70 7.73 7.78 7.79
0.500 7.60 7.69 7.74 7.72
0.250 7.74 7.92 7.81 7.79
Solvent Control 7.78 7.94 7.76 7.74
Control 7.67 7.84 7.82 7.78

Dissolved oxygen:

Water Quality Parameters at 0 h (Fresh Media)
(measured in one additional replicate per concentration level, solvent control and control)
Nominal
Test Item Concentration
[mg/L] Dissolved
O2-Concentration
[mg/L]
8.00 9.69
4.00 9.67
2.00 9.65
1.00 9.58
0.500 9.61
0.250 9.52
Solvent Control 9.53
Control 9.59

Water Quality Parameters after 48 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
8.00 4.12 4.08 3.06 3.40
4.00 4.55 4.44 4.21 3.54
2.00 6.12 6.24 6.06 5.58
1.00 6.59 6.92 6.74 6.80
0.500 7.30 7.33 7.47 7.02
0.250 8.06 7.49 7.30 7.64
Solvent Control 8.15 8.20 8.17 8.09
Control 8.14 8.12 8.24 8.18

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Natural river water of the river Innerste was used. This river is located near 31157 Sarstedt-Ruthe, Germany (52.25°, 9.83°). Specifications of the natural river are given in the table below. The natural river water was stored at 15 ± 2 °C until use in the test.

River Innerste
Location D-31157 Sarstedt-Ruthe
(Latitude 52.25°, Longitude 9.83°)
Sampling Date 2011-09-15
Weather on day of sampling Sunny, slightly cloudy, approximately 18 °C
Colour Yellowish to brownish, clear
pH 8.02
Conductivity [µS/cm] 960
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

- Culture medium different from test medium: No
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study: none
Immobilization Rates in the non GLP Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
10.0* 100 100 100 100 100 100
2.50* 0 0 0 10 10 10
0.625* 0 0 0 40 20 30
Solvent control* 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 3.68-4.35 mg/L

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 : 1.79 mg/L (CI 1.56 - 2.02 mg/L)

Reported statistics and error estimates:

EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The concentration effect relationships are shown graphically.
EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were estimated directly from the observation data, without mathematical calculation.

Any other information on results incl. tables

Immobilization Rates after 24 and 48 h of Exposure in the Definitive Test

                 (n = 20, divided into 4 replicates with 5 daphnids each)

	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
8.00*	100	100	100	100	100	100	100	100	100	100
4.00*	0	20	0	0	5	60	40	20	80	50
2.00*	0	0	0	0	0	0	20	0	0	5
1.00*	0	0	0	0	0	0	0	0	0	0
0.500*	0	0	0	0	0	0	0	0	20	5
0.250*	0	0	0	0	0	0	20	0	0	5
Solvent control*	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Naphthenic acids, reaction products with diethylenetriamine the 48 h- EC50 for Daphnia magna was estimated to be 4.00 mg/L (95 % confidence limits: 3.68 – 4.35 mg/L). The NOEC after 48 h was 2.00 mg/L. The LOEC after 48 h was 4.00 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Naphthenic acids, reaction products with diethylenetriamine(batch number: ESD0011831) were determined according to OECD 202 (2004) from 2012-07-18 to 2012-07-24, with the definitive exposure phase from 2012-07-18 to 2012-07-20, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h with 6concentrations of the test item Naphthenic acids, reaction products with diethylenetriaminein the range of 0.250 to 8.00 mg/L, prepared in natural river water in a geometric series with a dilution factor of 2using methanol as solvent. Therefore, a solvent control containing dilution water spiked with 0.1 mL methanol/L was tested additionally. Twenty daphnids were exposed to each concentration level, the solvent control and the control.

The concentrations of the test item Naphthenic acids, reaction products with diethylenetriamine wereanalytically verified by LC-MS/MS in the fresh media (0 h) and in the old media (48 h) of all concentration levels, the solvent control and the control.

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. Nevertheless, the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed). Due to the properties of the test item, nominal concentrations have to be used for assessment of the endpoints instead of measured ones. Sorption of the test item to the glassware of the test system was monitored.

The measured concentrations of the test item were in the range of 83 to 106 % of the nominal values in the fresh media (0 h) and 61 to 93 % in the old media (48 h).

Adsorption to glass walls of the test vessels was determined exemplarily at the test concentration levels of 1.00, 2.00 and 4.00 mg/L in separate replicates without daphnids (see Table 8). The amount of the test item adsorbed to the glass walls of the separate test vessels without daphnids after an exposure of 24 h was in the range of 10 to 16 % of the applied amount.

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

(based on the nominal test item concentrations)

Naphthenic acids, reaction products with diethylenetriamine
Endpoints (with 95 % confidence limits)	Test Duration [h]	Nominal Concentration of the Test Item [mg/L]
EC10	24	4.45 (CI: 4.07 – 5.30)
	48	2.60 (CI: 1.64 – 2.98)
EC50	24	6.00 (CI: 5.41 – 6.60)
	48	4.00 (CI: 3.68 – 4.35)
EC100	24	8.00
	48	8.00
NOEC	48	2.00
LOEC	48	4.00