Fatty acids, C18 unsat, reaction products with diethylenetriamine

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-31 to 2020-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The limit test item concentration and the controls were confirmed by analytical verification via LC-MS/MS on day -2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each. Determination of the test item was performed for the sediment, pore water and the overlying water in the controls and limit test item concentration under GLP. Except for the samples of day -2 and 0 all replicates contained worms. The method was implemented under non-GLP and documented finally in the GLP raw data.

Vehicle:

yes

Remarks:

Acetone

Details on sediment and application:

Sediment
Artificial sediment containing:
- 4 % peat, air-dried and finely ground
- 20 % kaolin, with a kaolinite content > 30 %
- 76 % quartz sand (> 50 % particles sized 50-200 µm)
- 0.2 % calcium carbonate (CaCO3) to achieve a pH of 6.92
The organic carbon content of the final mixture was 2.35 % (external determination, nonGLP).

Application
The respective test item amount was weighed out for the limit test item concentration and dissolved in acetone.
The appropriate amount of spiking solution (2.5 mL per replicate) was thoroughly mixed with a sub-quantity of the artificial sediment (10 g per replicate). After complete evaporation of the solvent, the spiked portion of artificial sediment was thoroughly mixed
with the remaining sediment. Demineralised water was added to the artificial sediment to adjust the humidity of the artificial sediment to a moisture of 30 %.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 45 g soil dry weight were filled into the test vessels.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test system Lumbriculus variegatus (Müller)
Lumbriculidae, Oligochaeta

Reason for the selection of the test system
Lumbriculus variegatus is a recommended species according to the guideline.

Origin
Bundesanstalt für Gewässerkunde (BfG), Am Mainzer Tor 1,
56068 Koblenz, Germany

Breeding
Breeding is performed at the test facility at 20  2 °C and diffuse light (100 - 500 lx, 16 h photoperiod daily).
The dissolved oxygen concentration is > 70 % of the air saturation value corresponding to 6.2 mg O2/L.
All worms used in the test originated from the same delivery of the supplier and the same stock. They were not used in another test.

Medium
Breeding of L. variegatus is performed on quartz sand in
reconstituted water containing 125 mL from each stock solution
poured in a can and filled up to 25 L with demineralised water.

Stock solution [g/L]
CaCl ∙ 2 H2O 55.80
MgSO4 ∙ 7 H2O 24.65
NaHCO3 12.95
KCl 1.15

Feeding TetraMin® flake food (TETRA) is provided twice per week.


The worms were synchronized 13 days prior to test start to minimize uncontrolled reproduction and regeneration.
At test start the worms had a body weight of 8.30 mg dry weight/10 worms (mean weight of two determinations).

Study type:

laboratory study

Test type:

static

Water media type:

other: Same as for holding. Overlying Test Water Properties at Day -2 pH-value Hardness [mgl/L CaCO3] Oxygen content [mg/L] Reconstituted Water used as Overlying Test Water 7.70 244 8.32

Type of sediment:

artificial sediment

Remarks:

Sediment Properties at Test Start Total Organic Carbon [%] 2.35* pH-value 6.92 Moisture [%] Sediment at test start 30 *) external measurement (2020-08-12 to 2020-08-14), non-GLP

Limit test:

yes

Duration:

28 d

Exposure phase:

total exposure duration

Remarks:

2020-09-02 to 2020-09-30

Hardness:

Overlying Test Water Properties at Day -2
Hardness
[mgl/L CaCO3]
Reconstituted Water used as Overlying Test Water 244



Water Parameters ( Hardness)
Day Parameter Control Solvent Control Nominal test item concentration
1000 mg test item/kg sediment dry weight
0
(before
insertion of worms) Total hardness [mg/L CaCO3] 291 301 313
28 Total hardness [mg/L CaCO3] 298 291 285

Test temperature:

Water Parameters (Temperature)
Day Parameter Control Solvent Control Nominal test item concentration
1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) Temperature [°C] 21.1 21.4 21.6
6 Temperature [°C] 20.0 19.8 19.8
13 Temperature [°C] 20.0 19.7 19.7
20 Temperature [°C] 19.8 19.2 19.3
28 Temperature [°C] 20.2 20.0 19.8

pH:

Overlying Test Water Properties at Day -2
pH-value
Reconstituted Water used as Overlying Test Water 7.70



Water Parameters (pH-value)
Day Parameter Control Solvent Control Nominal test item concentration
1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) pH 7.59 7.57 7.53
6 pH 7.81 7.77 7.75
13 pH 7.82 7.80 7.76
20 pH 7.84 7.70 7.69
28 pH 7.39 7.50 7.58

Dissolved oxygen:

Overlying Test Water Properties at Day -2
Oxygen content
[mg/L]
Reconstituted Water used as Overlying Test Water 8.32




Water Parameters (Oxygen-Concentration)
Day Parameter Control Solvent Control Nominal test item concentration 1000 mg test item/kg sediment dry weight
0
(before
insertion of worms) O2 [mg/L]* 8.12 8.22 8.09
6 O2 [mg/L]* 8.65 8.45 8.51
13 O2 [mg/L]* 8.63 8.46 8.64
20 O2 [mg/L]* 8.35 8.61 8.69
28 O2 [mg/L]* 8.48 8.66 8.70

*) 70 % of the air saturation value corresponds to 6.2 mg O2/L.

Ammonia:

Ammonium-N [mg/L]
Day after worm insertion Control Solvent Control mg test item/kg SDW
1000
0 0.35 < 0.05 0.14
2 0.11 0.53 0.49
5 < 0.05 0.09 0.33
7 0.52 0.30 1.55
9 0.42 0.43 1.54
12 0.09 0.36 1.17
14 0.15 0.09 0.68
16 0.21 0.15 0.37
19 0.26 0.14 1.16
21 0.48 0.15 1.07
23 0.35 0.16 0.92
26 0.34 0.51 1.32
28 0.43 0.23 1.06

Nominal and measured concentrations:

Nominal test item concentration:
limit test item concentration with 1000 mg/kg sediment dry weight

The measured concentration of the test item in the sediment was 114% at day 0, 87% at day 7 and 47% at day 28. Minor recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ.

Details on test conditions:

Environmental Conditions
Room temperature [°C] Photoperiod [h light] Light intensity [lx]
Nominal 20 ± 2 16 100 - 500
Actual 19 – 22 16 295 ± 56.7

Reference substance (positive control):

yes

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

total number of worms

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 100% active ingredient content

Details on results:

After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to the pooled control in the limit test item concentration. All validity criteria were fulfilled.

Mortality / Reproduction of Worms
No mortality of worms was observed in the controls and the limit test item concentration after exposure to the test item over 28 days .
The total number of worms and thus the reproduction did not significantly differ compared to the pooled control in the limit test item concentration.

Behaviour and Pathological Symptoms
No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in the limit test item concentration during the 28 days of exposure.

Dry Biomass of the Worms per Replicate
At test start the worms had a body weight of 8.30 mg dry weight/10 worms (mean weight of two determinations). At test end, the mean biomass dry weight of the worms was not statistically significantly different at the limit test item concentration compared to the pooled control.

Results with reference substance (positive control):

A reference item study is performed once a year with Potassium chloride. The most recent reference item test was carried out from 2020-06-22 to 2020-07-23 (Study-No. 72006IRS/RLA70116). The study was conducted with four reference item concentrations of 1.0 - 2.0 - 4.0 - 8.0 g/kg sediment dry weight.

Summary of all Effects and resulting NOEC, LOEC and EC50-values of the Reference Item Study
Effects Potassium Chloride
[g/kg sediment dry weight]
NOEC
Total worm number, Biomass 2

LOEC
Total worm number, Biomass 4

EC50 Total worm number
(CI 95%) 2.40 (1.99 – 2.86)

EC50 Biomass
(CI 95%) 2.61 (1.83 – 3.72)

CI = Confidence Interval

Reported statistics and error estimates:

NOEC/LOEC-values and statistical analyses

Since no statistically significant differences were observed between the control and the solvent
control, both controls were pooled for further evaluation.

Total worm number - Shapiro-Wilk’s Test on Normal Distribution
(reproduction) - Levene’s Test on Variance Homogeneity (with Residuals)
- Two-sample t-test Procedure

Biomass - Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Two-sample t-test Procedure

The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.


Software Calculations and graphics were carried out using software:
- Excel, MICROSOFT CORPORATION
- ToxRat Professional, TOXRAT SOLUTIONS GMBH
- Sigma Plot, SPSS INC.
All data presented in the tables were computer-generated and have been rounded for presentation. Thus, manual calculations of results based on the data in this report may yield in minor deviations from these figures.

Summary of all Effects, Based on Nominal Test Item Concentration

Effects	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	[mg/kg sediment dry weight]
LOEC (Total number of worms/Reproduction, Biomass)	>1000
NOEC (Total number of worms/Reproduction, Biomass)	≥1000

Mortality after 28 Days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Number of Dead or Missing						MV	SD ±	Mortality[%]
	1	2	3	4	5	6
Control	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	0	0
Pooled Control	-						0	0	0
1000	0	0	0	0	0	0	0	0	0

MV = Mean Value              SD = Standard Deviation

Total Worm Number after 28 Days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Number of Surviving							MV	SD ±	Inhibition to pooled control [%]^#	Sign.*
	1	2	3	4	5		6
Control	35	24	18	22	25		38	27.0	7.80	-	-
Solvent Control	18	36	29	37	18		24	27.0	8.44	-	-
Pooled Control	-							27.0	7.75	-	-
1000	28	23	16	28	19	15		21.5	5.75	20.4	No

*) = Two-sample t-test Procedure, alpha = 0.05       

MV = Mean Value       SD = Standard Deviation       #) = Negative value = Increase compared to pooled control

Reproduction after 28 days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Number of Reproduced							MV	SD ±	Inhibition to pooled control [%]^#	Sign.*
	1	2	3	4	5		6
Control	25	14	8	12	15		28	17.0	7.80	-	-
Solvent Control	8	26	19	27	8		14	17.0	8.44	-	-
Pooled Control	-							17.0	7.75	-	-
1000	18	13	6	18	9	5		11.5	5.75	32.4	N

MV = Mean Value       SD = Standard Deviation       *) =  t-test, alpha = 0.05

Worm Behaviour during 28 Days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Effect	Days after addition
		0	2	5	7	9	12	14	16	19	21	23	26	28
		Number of replicates (out of 6 or 4 replicates) showing particular feature
Control	ob	6	4	5	6	4	5	4	6	6	6	5	4	5
	a k	- -	2 -	1 -	- -	2 -	1 -	2 -	- -	- -	- -	1 1	2 -	1 -
	f	-	6	6	6	6	6	6	6	6	6	6	6	6
Solvent Control	ob	6	5	5	6	6	4	3	5	4	6	5	6	4
	a	-	1	1	-	-	2	3	1	2	-	1	-	2
	f	-	6	6	6	6	6	6	6	6	6	6	6	6
1000	ob a k f	6 - - -	4 2 - 6	5 1 - 6	6 - - 6	4 2 - 6	4 2 1 6	4 2 1 6	4 2 - 6	4 2 - 6	4 2 - 6	3 3 - 6	6 - - 6	6 - - 6

a) worm on sediment surface                       ob) no abnormality detected                  f) faeces on the sediment surface

k) worms stick together                            -) no observation

Worm Dry Biomass after 28 Days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Dry weight[mg] per replicate							MV	SD ±	Inhibition to control[%]^#	Sign.*
	1	2	3	4	5		6
Control	27.7	19.5	8.4	14.1	18.9		39.0	21.3	10.8	-	-
Solvent Control	9.7	30.0	26.5	37.6	9.7		10.0	20.6	12.4	-	-
Pooled Control	-							20.9	11.1	-	-
1000	23.9	19.9	11.1	11.3	10.3	10.2		14.5	5.9	31.0	N

MV = Mean value     SD = Standard deviation                    

*) Statistically significantly different compared to pooled control (Two-sample t-test Procedure)

Statistical Analyses

Threshold concentrations (NOEC) for Worm Number at 28 d

Comparison between Control and Solvent Control for Worm Number at 28 d

Shapiro-Wilk´s Test on Normal Distribution

Shapiro-Wilk´s Test on Normal Distribution with worm number at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.

      Treatm. [mg/kg]       Mean       s       n

      Control              27.0       7.80       6

      Solvent Control       27.0       8.44       6

Results:

Number of residuals = 14; Shapiro-Wilk´s W = 0.923; p(W) = 0.241; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.

Normality check was passed (Shapiro-Wilk´s; p > 0.01).

Variance homogeneity check (F-test) was passed (p > 0.01).

STUDENT-t test for Homogeneous Variances

STUDENT-t test for Homogeneous Variances with worm number at 28 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).

      Treatm. [mg/kg]       Mean       s       df       %MDD       t       p(t)       Sign.       p(F)

      Control        27.0       7.80                                          

      Solvent Control       27.0       8.44       10       38.7       0.00       1.000       -       0.867

+: significant; -: non-significant

There is no statistically significant difference between control and solvent control.

Statistical Characteristics of the Samples

Statistical characteristics with worm number at 28 d: Mean: arithmetic mean (X); Med: median; Min: minimum value. Max: maximum value; n: sample size; s: standard deviation; s%: coefficient of variation; s(X): standard error; %s(X): %standard error; 95%l. 95%u: lower. upper 95%-confidence limits.

      Treatm. [mg/kg]       Mean       Med       Min       Max       n       s       %s       s(X)       %s(X)       95%l       95%u

      Pooled Control       27.0       24.5       18.0       38.0       12       7.75       28.7       2.24       8.3       22.1       31.9

      1000.000       21.5       21.0       15.0       28.0       6       5.75       26.8       2.35       10.9       15.5       27.5

Shapiro-Wilk´s Test on Normal Distribution

Shapiro-Wilk´s Test on Normal Distribution with worm number at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.

      Treatm. [mg/kg]       Mean       s       n

      Pooled Control       27.0       7.75       12

      1000.000        21.5       5.75       6

Results:

Number of residuals = 14; Shapiro-Wilk´s W = 0.923; p(W) = 0.241; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.

Levene´s Test on Variance Homogeneity (with Residuals)

Levene´s Test on Variance Homogeneity (with Residuals) with worm number at 28 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance

      Source        SS       df       MSS        F        p(F)

      Treatment        13.444       1       13.444       1.415       0.252

      Residuals       152.000       16       9.500              

      Total        165.44        17                     

The Levene test indicates variance homogeneity (p > 0.010).

Variance homogeneity check was passed (p > 0.01).

Normal-distribution and variance-homogeneity requirements are fulfilled.

A parametric multiple test is advisable.

Two-sample t-test Procedure

Two-sample t-test Procedure with worm number at 28 d: Two-sample comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha  (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

      Treatm. [mg/kg]       Mean       s       df       %MDD       t       p(t)       Sign.       

Pooled Control              27.0       7.18                                          

      1000.000       21.5       7.18       16       -23.2       -1.53       0.073       -              +: significant; -: non-significant

There is no statistically significant difference between Pooled Control and 1000.000 mg/kg.

Threshold concentrations (NOEC) for Biomass after 28 days

Comparison between Control and Solvent Control for Biomass after 28 days

Shapiro-Wilk´s Test on Normal Distribution

Shapiro-Wilk´s Test on Normal Distribution with weight at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.

      Treatm. [mg/kg]       Mean       s       n

      Control       21.2667       10.78419       6

      Solvent Control       20.5833       12.34624       6

Results:

Number of residuals = 17; Shapiro-Wilk´s W = 0.938; p(W) = 0.292; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.

Normality check was passed (Shapiro-Wilk´s; p > 0.01).

Variance homogeneity ckeck (F-test) was passed (p > 0.01).

STUDENT-t test for Homogeneous Variances

STUDENT-t test for Homogeneous Variances with weight at 28 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).

      Treatm. [mg/kg]       Mean       s       df       %MDD       t       p(t)       Sign.       p(F)

      Control       21.2667       10.78419                                          

      Solvent Control       20.5833       12.34624       10       70.1       -0.10       0.921       -       0.774

+: significant; -: non-significant

There is no statistically significant difference between control and solvent control.

Shapiro-Wilk´s Test on Normal Distribution

Shapiro-Wilk´s Test on Normal Distribution with weight at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.

      Treatm. [mg/kg]       Mean       s       n

      Pooled Control       20.9250       11.05787       12

      1000.000       14.4500       5.92343       6

Results:

Number of residuals = 17; Shapiro-Wilk´s W = 0.941; p(W) = 0.330; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.

Levene´s Test on Variance Homogeneity (with Residuals)

Levene´s Test on Variance Homogeneity (with Residuals) with weight at 28 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance

      Source       SS       df       MSS       F       p(F)

      Treatment       77.293410       1       77.293410       3.858       0.067

      Residuals       320.593936       16       20.037121              

      Total       397.88735       17                     

The Levene test indicates variance homogeneity (p > 0.010).

Variance homogeneity check was passed (p > 0.01).

Normal-distribution and variance-homogeneity requirements are fulfilled.

A parametric multiple test is advisable.

Two-sample t-test Procedure

Two-sample t-test Procedure with weight at 28 d: Two-sample comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha  (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

      Treatm. [mg/kg]       Mean       s       df       %MDD       t       p(t)       Sign.       

      Pooled Control       20.9250       9.74833                                          

      1000.000       14.4500       9.74833       16       -40.7       -1.33       0.101       -       

+: significant; -: non-significant

There is no statistically significant difference between Pooled Control and 1000.000 mg/kg.

Peak areas related to measured concentrations

Measured peak areas and related concentrations may be different, because of daily calibration analysis

Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Artificial sediment and Sediment)

Nominal test item concetration [mg/kg SDW]	1000
Sampling date	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	Absolute Response			Calc. amount [µg/L]			Dilution factor
	Inj. 1	Inj. 2	Inj. 3	Inj. 1	Inj. 2	Inj. 3
-2	3573.974	3438.882	-	5.987	5.758	-	20000
0	2578.629	-	-	4.423	-	-	20000
7	1339.793	1353.962	-	3.479	3.511	-	20000
28	1141.606	1868.273	1804.823	2.589	4.124	3.990	10000

Calc.. amount              Calculated amount of the test item without weighing and dilution factors

Inj.              Injection

-       Not determined

Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Aqueous Layer)

Nominal test item concetration [mg/kg SDW]	1000
Sampling date	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	Absolute Response		Calc. amount [µg/L]		Dilution factor
	Inj. 1	Inj. 2	Inj. 1	Inj. 2
0	7187.447	6663.283	0.959	0.910	20
7	28760.076	27825.215	4.514	4.371	20
28	5347.490	6428.223	0.874	1.111	20

Calc.. amount              Calculated amount of the test item without weighing and dilution factors

Inj.              Injection

Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Pore Water)

Nominal test item concetration [mg/kg SDW]	1000
Sampling date	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	Absolute Response		Calc. amount [µg/L]		Dilution factor
	Inj. 1	Inj. 2	Inj. 1	Inj. 2
0	48551.934	50726.215	7.538	7.870	20
7	42772.543	41741.641	6.655	6.497	200
28	34391.086	35444.617	7.222	7.452	200

Calc.. amount              Calculated amount of the test item without weighing and dilution factors

Inj.              Injection

Measured Concentrations and Percent of Nominal of Fatty acids, C18 unsat, reaction products with diethylenetriamine (artificial sediment) for Day -2

Sampling date	Day -2
Artificial sediment used for preparation of the test item concentration [mg/kg]	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	Meas. conc. [mg/kg]	%
1000	1170	117
Solvent Control	< LOQ
Control	< LOQ

Meas. conc.       = Measured concentration (dilution factor taken into account)

%                     = Percent of the nominal concentration of the test item of the quartz sand

LOQ               = 100 mg/kg of the test item

Measured Concentrations and Recovery Rates of Test Item (Aqueous Layer, Pore Water and Sediment) - Day 0       

Nominal test item concentration [mg/kg SDW]		1000	Control	Solvent Control
Amount applied per test vessel [mg]^1)		45.0
Aqueous Layer	Meas. amount [mg]	< LOQ	< LOQ	< LOQ
	%
Pore Water	Meas. amount [mg]	0.000585	< LOQ	< LOQ
	%	0
Sediment	Meas. amount [mg]	51.5	< LOQ	< LOQ
	%	114
Total Amount	[mg]	51.5	< LOQ	< LOQ

1)       = Based on 45 g sediment (DW) and 150 mL dilution water

Meas. amount       = Measured amount, dilution and weighing factor taken into account

%       = Percent of the nominal concentration of the test item

LOQ       = Limit of quantification of the analytical method (sediment: 100 mg test item/kg, aqueous layer: 100 µg test item/L)

Measured Concentrations and Recovery Rates of Test Item (Aqueous Layer, Pore Water and Sediment) - Day 7 and 28       

Day 7
Nominal test item concentration [mg/kg SDW]		1000	Control	Solvent Control
Amount applied per test vessel [mg]^1)		45.0
Aqueous Layer	Meas. amount [mg]	0.130	< LOQ	< LOQ
	%	0
Pore Water	Meas. amount [mg]	0.00516	< LOQ	< LOQ
	%	0
Sediment	Meas. amount [mg]	39.2	< LOQ	< LOQ
	%	87
Total Amount	[mg]	39.3	< LOQ	< LOQ
	%	87
Day 28
Nominal test item concentration [mg/kg SDW]		1000	Control	Solvent Control
Amount applied per test vessel [mg]^1)		45.0
Aqueous Layer	Meas. amount [mg]	0.00330	< LOQ	< LOQ
	%	0
Pore Water	Meas. amount [mg]	0.00629	< LOQ	< LOQ
	%	0
Sediment	Meas. amount [mg]	21.3	< LOQ	< LOQ
	%	47
Total Amount	[mg]	21.3	< LOQ	< LOQ

1)       = Based on 45 g sediment (DW) and 150 mL dilution water

Meas. amount       = Measured amount, dilution and weighing factor taken into account

%       = Percent of the nominal concentration of the test item

LOQ       = Limit of quantification of the analytical method (sediment: 100 mg test item/kg, aqueous layer: 100 µg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to the pooled control in the limit test item concentration.
All validity criteria were fulfilled.

Executive summary:

Summary

The effects of the test item FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE, Batch number 1865159) on the endobenthic oligochaete Lumbriculus variegatus in a water-sediment system were determined at the test facility in Sarstedt, Germany, from 2020-08-31 to 2020-10-09, with the definitive exposure phase from 2020-09-02 to 2020-09-30. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the limit test item concentration 1000 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial soil treated with the solvent were set up. Six replicates per control and limit test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O2-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentration, each.

Analytical evaluation of the test item FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE and controls was carried out via LC-MS/MS on day -2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each.

The measured concentration of the freshly spiked artificial sediment on day -2 was 117 %, thus indicating its proper preparation. The control samples were < LOQ. After 7 and 28 days 87 and 47% were recovered respectively.

The pore water concentration in these samples remained relatively constant

After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.

The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to pooled control in the limit test item concentration.

All validity criteria were fulfilled.

Summary of all Effects, Based on Nominal Test Item Concentration

Effects	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	[mg/kg sediment dry weight]
LOEC (Total number of worms/Reproduction, Biomass)	>1000
NOEC (Total number of worms/Reproduction, Biomass)	≥1000

Description of key information

There is one long term sediment test with lumbriculus variegatus available which was performed according to OECD 225 under GLP conditions and fulfilled all validity criteria.

The test was performed as limit test at 1000 mg a.i./kg dw and no effects were observed.

In addition to measured data the PNECsed was calculated using the equilibrium partitioning method (EPM).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

1 000 mg/kg sediment dw

Additional information

The effects of Fatty acids, C18 unsat, reaction products with diethylenetriamine (AAI-DETA) on the endobenthic oligochaete Lumbriculus variegatus in a water-sediment system were determined under GLP conditions. The study was formed according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the limit test item concentration 1000 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial sediment treated with the solvent were set up. Six replicates per control and limit test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O₂-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentration, each.

Analytical evaluation of the test item Fatty acids, C18 unsat, reaction products with diethylenetriamine and controls was carried out via LC-MS/MS on day -2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each. The measured concentration of the freshly spiked artificial sediment on day -2 was 117 %, thus indicating its proper preparation. The control samples were < LOQ. 

After 28 days of exposure, Fatty acids, C18 unsat, reaction products with diethylenetriamine induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.

The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to pooled control in the limit test item concentration. For further endpoints, please see Table 1 below. 

All validity criteria were fulfilled.

 

Table 1: Summary of all Effects, Based on Nominal Test Item Concentration

Effects	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	[mg/kg sediment dry weight]
LOEC (Total number of worms/Reproduction, Biomass)	> 1000
NOEC (Total number of worms/Reproduction, Biomass)	≥ 1000

 

The PNECsed of 10 mg/kg dw is according to REACH guidance R.10 calculated from the available long-term lumbriculus test by applying the default safety factor of 100.

In the absence of any measured toxicity to sediment organisms the PNECsed can also be calculated using the Equilibrium Partitioning Method (EPM). Because of the availability of a long-term sediment test for a substance for which it is indicated that the use of EPM to predict the PNECsediment is not appropriate (surfactant, ionic). The PNECsed of 10 mg/kg dw will be used for risk assessment.

 

For comparison reasons the PNECsed was also calculated using the Equilibrium partitioning method (EPM). According to REACH guidance R.10 this method uses the PNEC_aquatic and the sediment/water partitioning coefficient as inputs.

The dose response results for the algae test are calculated based on nominal Test Loadings. The PNECaquatic is based on the Algae E_rL₁₀ after 72 hours is 20.7 µg/L. The EL10 for daphnia was observed to be 133 µg/L.

The PNEC_sed is then calculated using the equations detailed in the TGD:

 

               PNEC_sed                         =   K_susp-water* PNEC_aquatic * 1000 * 1 / RHO_susp

Where:        PNEC_aquatic                                =      2.07 µg/L

                    K_susp-water                            =      23500.9 m³.m^-3

                    RHO_susp                             =      1150 kg.m^-3(TGD, equ. 18)

                    PNEC_sed-EPM                      =      42.3 mg/kg ww   =  194.6 mg/kg dw

                Applying the additional safety factor for exposure via ingestion: 10

PNEC_sed-EPM                     = 4.23 mg/kg ww

                                            = 19.46 mg/kg dw