Copepod Tisbe battagliai Shrimp Atyaephyra desmarestii · 2020. 9. 1. · Tisbe battagliai:Lethal and reproductive endpoints. Ecotoxicology and Environmental Safety 72 (7), 1881-1886.

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Toxicity testing and behavioral changes in two species exposure to several pharmaceutical

compounds :Copepod Tisbe battagliai Shrimp Atyaephyra desmarestii

Nieto,E1.,Drake,P1.,Trombini1,C., González-Ortegón, E. Hampel, M1.,Blasco,J1.1Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC)

Pharmaceuticals used in the bioassays

Carbamazepine (CA) anticonvulsant, used to treat epilepsy.

Atenolol (AT) Beta-blockers affect the heart and circulation,used to treat angina and hypertension.

Acetaminophen (AC) Analgesic and antipyretic, pain relieve and reduction of fever.

Diclofenac (DF) used to treat pain, inflammatory disorders.

Ibuprofen (IB) nonsteroidal anti-inflammatory, used to reduce fever and treat pain or inflammation.

Objectives

Single and mixture toxicity of several compounds selected individually.

Ecological risk assessment (PEC/PNEC)

Acute toxicity evaluation of several compounds selected individually.

Assessment of chronic effects of individual compounds:

Feeding behaviour.Osmoregulation.Respiration rates.

Ecological risk assessment (PEC/PNEC)

Test organisms: Tisbe battagliai

• Marine, estuarine copepod Tisbe battagliai (Crustacea, Copepoda, Harpacticoida) • Distribution: shallow waters of coastal regions of Europe and US Atlantic coast.• Feeding: epiphytic micro-algae, detritus and bacteria.• Development:

Adult female carrying ovaSecond stage copepodid

Lethal endpoints in copepod

Toxicity bioassay (Diz et al. 2009)

•

Acute toxicity test Ecological risk assessment

Lethal concentration and 95% confidence limits of the substances tested

Substance LC5(mg/L) LC10(mg/L) LC20(mg/L) LC50(mg/L) PEC(µg/L) Reference

PNEC

PEC/PNEC RiskLC50(96h)

AF=1000

Acetaminophen

6,7 13,4 33,5 67,8

10 (Kolpin et al.,2002) 0,068 0,14 Lown.D (-13-28,4) (15,0-44,8) (58,2-77,8)

Carbamazepine

4,7 9,4 18,8 59

5 [Blasco & Del Vall, 2008] 0,059 0,08 Lown.D n.D (3,7-31,0) (45,6-80)

Diclofenac

2,8 4,5 6,3 9,5

0,8 [Blasco & Del Vall, 2008] 0,01 0,08 Low(-0,4-4,7) (2,0-6,1) (4,4-7,8) (8-11,3)

Ibuprofen

5,65 11,3 25,5 49,7

10 [Blasco & Del Vall, 2008] 0,05 0,2 Lown.D (-10,9-24,7) (8,4-36,7) (38,8-59,8)

Mixture toxicityAC-CA

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC5 /2

LC10 /2

LC20 /2

LC50 /2

LC80 /2

AC-DF

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC5 /2

LC10 /2

LC20 /2

LC50 /2

LC80 /2

AC-IB

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC10 /2

LC20 /2

LC50 /2

LC80 /2

CA-DF

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC5 /2

LC10 /2

LC20 /2

LC50 /2

LC80 /2

CA-IB

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC5 /2

LC10 /2

LC20 /2

LC50 /2

LC80 /2

DF-IB

Concentration (LCx/2)

% M

orta

lity

0

20

40

60

80

100

120

Controls

LC5 /2

LC10 /2

LC20 /2

LC50 /2

LC80 /2

Measured toxicity of binary mixture of Acetaminophen (AC), Carbamazepine (CA), Diclofenac (DF) and Ibuprofen (IB) in comparison to the singly measured toxicities and the mixture toxicity predicted by concentration addition. 

DISCUSSION

All selected pharmaceutical compounds produced mortality in nauplii.Diclofenac seems to be the most toxic compound.

Mixture mortality is much higher than the effect of individual compounds as well as the expected effects calculated using concentration addition.

Tested concentrations are 10.000 to 100.000 fold higher than concentration detected in the aquatic environment for all compounds.

Observed toxicity for mixtures of two compounds was somewhat stronger than expected from very weak effects measured singly.

Mixtures including Diclofenac seem to be the most toxic, particularly AC-DF and IB-DF (mortality ≥80% at low concentrations LC5/2)

Classification of tested compounds according to the LC50 values(CEC, 1996):•Diclofenac: “toxic to aquatic organisms” (LC50 =1-10 mgL-1). • Acetaminophen, Carbamazepine and Ibuprofen: “harmful” (LC50 =11-100 mgL-1).

•Test organisms: Atyaephyra desmarestii (Millet,1831)

• Freshwater shrimp (Crustacea, Decapoda, Atyidae).• Distribution: Mediterranean species (Tittizer et al., 2000), has now spread to Central European rivers

flowing into the Baltic, Northern and Black Sea due to vessel traffic (Tittizer et al., 1996).• Feeding: phytophylic species, takes place in calm waters, but well oxygenated, with good presence of

macrophytes, food is detritivores, coming to eat algae, mud or fecal pellets (Fidalgo & Gerhardt, 2002).

Location of sampling point

Sierra de Grazalema

Embalse de Zahara de la Sierra

Rio Guadalete

Punto de muestreo

Material & Methods

Semi-static exposure system

Acute Toxicity Test

Method EPA OPPTS 850.1015/1020LC50 Determination - 96 hours.Testing in glass jars.5 glass jars with concentrations and 1 jar test control all in duplicate.FW as a means of exposureMedium renewal every 48 hours.10 individuals per jars.Variables to be monitored daily:

- Dissolved oxygen concentration- pH- T ª

Photoperiod (16 hours light and 8 of darkness).Removing dead bodies daily and moults.Sampling of water exposure for analysis of nitrogen compounds.At the end of the experiment the survivors were frozen in liquid nitrogen for enzymatic analysis of responses.LC50 values were estimated by adjusting the program GLMstat.

Lethal endpoints

Acute toxicity test Ecological risk assessment

Lethal concentration and 95% confidence limits of the substances tested

Substance LC5(mg/L) LC10(mg/L) LC20(mg/L) LC50(mg/L) PEC(µg/L) ReferencePNEC

PEC/PNEC

RiskLC50(96)

AF=1000

Carbamazepine36,6 49,7 63,9 88,2

5[Blasco & Del

Vall.,2008] 0,088 0,06 Low(2,9-54,8) (22-65,4) (42,3-77,4) (74,2-100,6)

Diclofenac0,1 1,9 3,9 7,3

0,8[Blasco & Del

Vall.,2008] 0,007 0,11 Low(-3,2-1,8) (-0,5-3,3) (2,3-5,0) (6,2-9)

Ibuprofen5,4 6,5 7,7 9,7

10[Blasco & Del

Vall.,2008] 0,01 1,03 Medium(0,3-7,1) (2,6-7,9) (5,0-8,9) (8,4-11,3)

Atenolol14,3 101,3 195,7 357,1

0,89[Blasco & Del

Vall.,2008] 0,357 0,002 Low(-151,6-110,8) (-31,1-182,7) (95,7-264,9) (290,3-427,3)

Sub-lethal endpoints

Efecct on feeding

* 10 individuals per compound.

* 10 control subjects solvent.* 1 individual per glass.* 10 days of exposure.* 1 day of acclimatization.* Not food 24 hours before exposure.* Semi static conditions.* Food with TetraMin .* Reading for 300 minute setting daily every .* Reading the rate of filling of the digestive tract.* Ecological concentration were used:

* CA – 10 µg/L* DF – 10 µg/L* IB – 25 µg/L* AT – 10 µg/L

Results & discussion

Ecological concentrationS= 0‰Tª= 21ºC10 days exposed

C CA DF AT IB

Hem

olyp

h os

mol

ality

(mm

ol k

g-1 )

300

320

340

360

380

400

420

440

Osmoregulation rates

Ecological concentrationS= 0‰Tª= 20ºC9 days exposed

Ingestion rates

W a te r o xyg e n co n ce n tra tio n (m g l-1)

0 1 2 3 4 5 6

Rel

ativ

e sp

ecifi

c co

nsup

tion

rate

0

2 0

4 0

6 0

8 0

1 0 0O b s e rv e d c o n s u m p tio n ra teN o n o o xyg e n -re g u la tin g ra teP e rfe c to x yg e n -re g u la tin g ra te

Ecological concentrationS= 0‰Tª= 20ºC6 days exposed4 days respiration measuresRespiration rates corrected for weight of shrimp.

DISCUSSION

Diclofenac is the most toxic and Atenolol the least toxic compound in acute toxicity test.

Classification of tested compounds according to the LC50 values(CEC, 1996):Diclofenac: “toxic to aquatic organisms” (LC50 =1-10 mgL-1). Carbamazepine and Ibuprofen: “harmful” (LC50 =11-100 mgL-1).

According Dunnet test no significant differences at 30, 60 and 90 minutes between control and treatment in the feeding test to 20ºC.

According Dunnet test no significant differences between control and treatment in the Osmoregulation test to 20ºC.

Respiration rates at concentrations of 5 mg O2 /L (oxygenated) and 3 mg O2 /L(moderate hypoxia) no significant differences between control and treatment to 20ºC.

Respiration rates at concentrations of 1 mg O2 /L (severe hypoxia) Diclofenac show a respiration rates lower than the control but is not very significant.

The degree of oxy-independence is not significant at any level of oxygen concentration to 20 º C.

CONCLUSIONS

T. battagliai and A.desmarestii are a suitables candidates for the evaluation of effects of pharmaceuticals compounds.

The ecological risk assessment through of ratio PEC/PNEC show low risk levels ( “low risk”from 0.01 to 0.1) associated of the occurrence of these compounds in STPs effluents, only the ibuprofen present s “medium risk” from 0.1 to 1 when exposed to the organisms A.desmarestii

References:• Diz F.R.,Araujo C.V.M.,Moreno-Garrido I., Hampel M., Blasco J.(2009). Short-term toxicity test on the harpacticoid copepod

Tisbe battagliai: Lethal and reproductive endpoints. Ecotoxicology and Environmental Safety 72 (7), 1881-1886.

• Cleuvers, M. (2003). Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects. Toxicology Letters 142 (2003) 185-194.

• Gonzalez-Ortegón E., Cuesta J.A., Pascual E., Drake P.(2009). Assessment of the interaction between the white shrimp, Palaemon longirostris, and the exotic oriental shrimp, Palaemon macrodactylus, in a European estuary (SW Spain). Biol Invasions (2010) 12:1731–1745. (DOI 10.1007/s10530-009-9585-2).

• Gonzalez-Ortegon E., Cuesta J.A., Pascual E., Drake P. (2006). Field distribution and osmoregulatory capacity of shrimps in a temperate European estuary (SW Spain). Estuarine, Coastal and Shelf Science 67 (2006) 293e302.

• Blasco J.,Del Valls A. (2008). Impact of Emergent Contaminants in the Environment: Environmental Risk Assessment. Emerging Contaminants from Industrial and Municipal Waste. Editors: D.Barcelo., M.Petrovic

• D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg and L.B. Barber, et al. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: a national reconnaissance. Environ Sci Technol, 36 (2002), pp. 1202–1211.

• EC,1996, Techinical Guidance Documents in Support of the Commission Directive 93/667/EEC on risk assessment for new notified substances an the Commission regulation (EC) 1488/94 on Risk substances, European Chemical Bureau, Ispra Italy, 19th April 1996, part 1,2 and 3.

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