Naphthenic acids, reaction products with diethylenetriamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-13 to 2012-08-17

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 201 (Alga, Growth Inhibition 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: 0.102 - 0.256 - 0.640 - 1.60 - 4.00 - 10.0 mg/L
- Sampling method: The concentrations of the test item were analytically verified from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken directly from freshly prepared test concentrations prepared with algae, except the control and solvent control. At the end of the test, samples were taken directly from the pooled test replicates.
The sorption of the test item on glass was quantified. Separate replicates of the three highest concentration levels without algae were prepared. After sampling for test medium analysis (aqueous phase), the glassware was emptied and rinsed carefully with demineralised water to remove all test item dissolved in remaining test media. Thereafter the vessels were rinsed with 25 mL of acetonitrile containing 2 % formic acid. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration.
Additionally sorption to the walls of the glass container was checked out of two test replicates (prepared with algae) of a concentration level close to the ErC50- and EyC50-value.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Test solutions

Vehicle:

no

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 200 g/L was prepared in a measuring flask (10 mL capacity) by directly weighing of 2000 mg test item into the measuring glass flask and filling up with methanol to 10 mL thereafter.
Further stock solutions were prepared out of this stock solution by diluting with methanol. Appropriate aliquots of the stock solution of 200 g/L (as specified in the table below) were taken and pipetted directly into measuring glass flasks (5 mL capacity) 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 / test concentrations.
The test concentrations were freshly prepared at the beginning of the exposure phase (0 h) by adding 0.02 mL stock solution to 400 mL of dilution water. The test concentrations were homogenised by agitation before use.
- Eluate: Natural River water
- Differential loading: 0.102 - 0.256 - 0.640 - 1.60 - 4.00 - 10.0 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.3 °C, Min: 21.5 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
10.0 8.28 8.16
4.00 8.27 8.14
1.60 8.27 8.12
0.640 8.24 8.13
0.256 8.20 8.17
0.102 8.14 8.18
Solvent control 8.13 8.23
Control 8.10 8.33

Dissolved oxygen:

Not measured

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: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4332 cells/mL
- Control end cells density: Mean 1057862 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline (OECD)
Water Parameter of the River Water
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

Composition of Dilution water (OECD)
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test without Methanol (0-72 h)
Nominal test item concentration
[mg/L] Growth rate inhibition [%] Yield inhibition [%]
10 94 100
1 27 76
0.1 - 1 - 7
0.01 0 - 1

Results of the Range Finding Test with Methanol (0-72 h)

Nominal test item concentration
[mg/L] Growth rate inhibition [%] Yield inhibition [%]
10 88 100
1 32 82
0.1 18 61
0.01 14 52
0.001 15 54
Solvent control
(0.1 mL methanol/L) 15 54

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 1.33-1.69 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 9.38-0.66 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The stock solution was slightly turbid. After 24 hours a concentration-related agglutination of the test item was observed in the two highest concentration levels.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.540 (CI 0.528 - 0.551) mg/L
Yield Inhibition: 0.324 (CI 0.304 - 0.347) mg/L

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield after 72 hours. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. The normality test failed for the yield data. No eligible transformation method was found for these data.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals
EC10-, EC20- and EC50-values were carried out using standard procedures.

Any other information on results incl. tables

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
10.0		1		4332		8712	LOQ	LOQ
		2		4332		9750	3863	LOQ
		3		4332		10697	5690	2762
		Mean		4332		9720	n.a.	n.a.
4.00		1		4332		9351	4998	2504
		2		4332		11084	6347	4284
		3		4332		10521	7627	5693
		Mean		4332		10319	6324	4160
1.60		1		4332		16141	33539	64552
		2		4332		12892	52321	56274
		3		4332		15242	35011	74025
		Mean		4332		14758	40290	64950
0.640		1		4332		19031	90272	241729
		2		4332		23073	96310	229161
		3		4332		19585	100613	274298
		Mean		4332		20563	95732	248396
0.256		1		4332		19645	145378	675399
		2		4332		20236	137585	712447
		3		4332		19082	134189	738299
		Mean		4332		19654	139051	708715
0.102		1		4332		17616	157109	825793
		2		4332		22610	159764	826233
		3		4332		21016	159745	800947
		Mean		4332		20414	158873	817658
Solvent control		1		4332		21333	152562	747105
		2		4332		19157	158921	824472
		3		4332		17188	150209	745312
		4		4332		22060	155380	752514
		5		4332		18729	128421	602750
		6		4332		21063	136321	780411
		Mean		4332		19922	146969	742094
Control		1		4332		17638	157946	1017135
		2		4332		18500	148284	1058083
		3		4332		18971	169343	960902
		4		4332		18182	154103	1076449
		5		4332		16842	166362	1113843
		6		4332		21733	185049	1120762
		Mean		4332		18644	163515	1057862

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		-0.150	100		-1570	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
4.00	1		-0.183	100		-1828	100
	2		-0.004	100		-48	100
	3		0.091	94.7		1361	99.8
	Mean	(+)	-0.032	98.2	(+)	-172	99.9
1.60	1		0.900	47.4		60220	91.8
	2		0.855	50.1		51942	93.0
	3		0.946	44.8		69693	90.6
	Mean	(+)	0.900	47.4	(+)	60618	91.8
0.640	1		1.34	21.7		237397	67.8
	2		1.32	22.8		224829	69.5
	3		1.38	19.3		269966	63.4
	Mean	(+)	1.35	21.3	(+)	244064	66.9
0.256	1		1.68	1.74		671067	9.04
	2		1.70	0.70		708115	4.02
	3		1.71	0.01		733967	0.51
	Mean	(-)	1.70	0.82	(-)	704383	4.52
0.102	1		1.75	-2.17		821461	-11.3
	2		1.75	-2.18		821901	-11.4
	3		1.74	-1.57		796615	-7.98
	Mean	(-)	1.75	-1.97	(-)	813326	-10.2
Solvent control	1		1.72			742773
	2		1.75			820140
	3		1.72			740980
	4		1.72			748182
	5		1.65			598418
	6		1.73			776079
	Mean		1.71			737762

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Naphthenic acids, reaction products with diethylenetriamine was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for the inhibition of the growth rate and the inhibition of yield after 72 hours, were 0.256 mg/L for both endpoints. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours, were 0.640 mg/L for both endpoints. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 1.50 (1.33 – 1.69) and 0.511 (0.485 – 0.537) mg/L, respectively.
All effect levels are given based on nominal concentrations of Naphthenic acids, reaction products with diethylenetriamine.

Executive summary:

The toxicity of Naphthenic acids, reaction products with diethylenetriamine (batch number: ESD0011831)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2012-08-13 to 2012-08-17. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4332 cells/mL. Based on a preliminary test 6 concentration levels were tested in a geometrical series with a dilution factor of 2.5: 0.102 - 0.256 - 0.640 - 1.60 - 4.00 - 10.0 mg/L using methanol as solvent. Therefore, a solvent control containing dilution water spiked with 0.05 mL MeOH/L was tested additionally. Three replicates were tested for each test item concentration and six replicates for the solvent control and the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Naphthenic acids, reaction products with diethylenetriaminewere analytically verified by LC-MS/MS at test start and test end of all test concentrations and the control groups. Four precursor ions representative for the present mass ranges were chosen for the analysis. The measured concentrations for Naphthenic acids, reaction products with diethylenetriamine in freshly prepared media were in the range of 68 – 102 % of the nominal values. In the old media the test item gave recoveries of 60 – 101 % of the nominal values. Adsorption of the test item to the test vessels after 72 h of exposure was determined from the test concentrations framing the EC₅₀ at the test concentration levels of 0.640 and 1.60 mg/L of test vessels with algae and 1.60, 4.00 and 10.0 mg/L in separate replicates without algae. The adsorbed amount of the test item in vessels without algae was in the range of 16 to 21 % of the applied amount, in vessels with algae at 4 – 7 %.

The test item Naphthenic acids, reaction products with diethylenetriamine 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 from such tests depend from 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 (see Table 1).

NOEC, LOEC, EC - values and 95 % Confidence Intervals of
Naphthenic acids, reaction products with diethylenetriamine (0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Growth Rate Inhibition
NOEC	0.256
LOEC	0.640
ErC10	0.506 (0.376 – 0.661)
ErC20	0.754 (0.613 – 0.907)
ErC50	1.50 (1.33 – 1.69)
	Inhibition of Yield
NOEC	0.256
LOEC	0.640
EyC10	0.284 (0.262 – 0.307)
EyC20	0.339 (0.316 – 0.362)
EyC50	0.511 (0.485 – 0.537)